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 (K i ) in the micromolar range and a high-affinity site with a constant K i 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.