Human corneal endothelial cell expression of Na+,K+-adenosine triphosphatase isoforms.
OBJECTIVE
To determine the expression of alpha subunits and different isozymes of Na+,K+-adenosine triphosphatase (ATPase) in human corneal endothelial cells (HCECs).


METHODS
Immunoblot and RNA analysis of Na+,K+-ATPase alpha subunit expression were performed in preparations from HCECs that had been immortalized by transformation with simian virus 40. Na+,K+-ATPase activity was determined by constructing dose-response curves for the ouabain inhibition of Na+,K+-ATPase activity in human corneal endothelial cells.


RESULTS
Both messenger RNA analysis and immunoblot studies indicated that HCECs express ATPase catalytic alpha1 and alpha3, but not alpha2 and alpha4, subunits. A limited amount of alpha3 subunit was expressed in HCECs compared with the alpha1 subunit. Biochemical analyses of Na+,K+-ATPase activity revealed 2 independently active Na+,K+-ATPase isoenzymes, a low-affinity site with a kinetic parameter for ouabain inhibition constant (Ki) in the micromolar range and a high-affinity site with a constant Ki in the nanomolar range. These 2 sites may be associated with alpha1 and alpha3 isoforms, respectively, expressed in HCECs.


CONCLUSIONS
Human corneal endothelial cells express alpha1 and alpha3 isoforms of Na+,K+-ATPase, and both polypeptides are catalytically competent in these cells. Defining the components of Na+,K+-ATPase in HCECs is an important step toward elucidating the mechanisms that regulate corneal endothelial ionic pump function as well as the pathogenesis of corneal diseases associated with corneal edema.
Huang
Bo
B
Pepose Vision Institute, Chesterfield, Mo 63017, USA.
Blanco
Gustavo
G
Mercer
Robert W
RW
Fleming
Tim
T
Pepose
Jay S
JS
D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000166:cytology / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes; D010042:Ouabain / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D013539:Simian virus 40; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D012333:RNA, Messenger; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase