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

Abstract

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.

Telomerase Immortalization of Human Corneal Endothelial Cells Yields Functional Hexagonal Monolayers

“…Both α and β subunits exist in different isoforms that are synthesized in a tissue-specific manner [27]. HCEn is known to distinctly express the α1 and α3 isoforms, which are responsible for hydrolyzing ATP and ion transportation [28]. Real-time PCR showed similar expression of the Na/K ATPase α1 subunit (Figure 5B) and upregulation of the Na/K ATPase α3 subunit (Figure 5C) in HCEnC-21 and HCEnC-21T cells compared to 21M primary cells.…”

Digoxin derivatives with selectivity for the α2β3 isoform of Na,K-ATPase potently reduce intraocular pressure

“…In principle, the ability of DMcP and DcB to reduce the basal IOP could be relevant not only to optical hypertension and primary open angle glaucoma but also to normotensive glaucoma for which reduction of IOP below the basal level is mandatory (1). Regarding local toxicity, α2β3 (or α2β2)-selective digoxin derivatives could have an additional benefit of producing only minimal side effects on corneal endothelium (23) or lens epithelium (24) because these tissues express mainly the ubiquitous α1 isoform. On the other hand, other ocular tissues such as retina express various combination of α1−3 and β1−3 isoforms (25), making it essential to examine possible local toxic effects of the digoxin derivatives.…”

Role of Insulin in Regulation of Na+-/K+-Dependent ATPase Activity and Pump Function in Corneal Endothelial Cells

“…52 The ␣ 2 isoform appears to be involved in regulating Ca 2ϩ transients involved in muscle contraction, whereas the ␣ 1 isoform probably plays a more generalized role. 52 Huang et al 53 reported that both the ␣ 1 and the ␣ 3 isoforms are expressed in human corneal endothelial cells. We examined Na,K-ATPase ␣ 1 -subunit expression in corneal endothelial cells because of its generality.…”