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Bo Huang, MD, PhD; Gustavo Blanco, PhD; Robert W. Mercer, PhD; Tim Fleming, PhD; Jay S. Pepose, MD, PhD

Arch Ophthalmol. 2003;121:840-845.

Objective  To determine the expression of 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 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 1 and 3, but not 2 and 4, subunits. A limited amount of 3 subunit was expressed in HCECs compared with the 1 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 1 and 3 isoforms, respectively, expressed in HCECs.

Conclusions  Human corneal endothelial cells express 1 and 3 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.

From the Pepose Vision Institute, Chesterfield, Mo (Drs Huang and Pepose); the Departments of Ophthalmology and Visual Sciences (Dr Pepose), Cell Biology and Physiology (Dr Mercer), and Surgery (Dr Fleming), Washington University School of Medicine, St Louis, Mo; and the Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City (Dr Blanco). The authors have no relevant financial interest in this article.

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