Human Corneal Endothelial Cell Expression of Na+, K+–Adenosine Triphosphatase Isoforms

OBJECTIVE To determine the expression of α 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 α 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 α1 and α3, but not α2 and α4, subunits. A limited amount of α3 subunit was expressed in HCECs compared with the α1 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 α1 and α3 isoforms, respectively, expressed in HCECs. CONCLUSIONS Human corneal endothelial cells express α1 and α3 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.Arch Ophthalmol.2003;121:840-845 -->