The Ca²⁺/ Nucleotide Nanocomplex formulation enhances the pump function of corneal endothelial cells by increasing the activity of Na⁺/K⁺ Dependent ATPase.

Abstract

Purpose : Na, K-ATPase exist in the membrane of the corneal endothelial cell and control the corneal hydration through the pump function. It also plays an important role in preserving the transparency of the cornea. In this study, we investigated the effect of Ca²⁺/ Nucleotide (NT) nanocomplex (NC) on Na, K-ATPase and the pump function of the corneal endothelial cells.

Methods : Bovine corneal endothelial cells were used, and the cells were treated with Ca²⁺/ Nucleotide (NT) nanocomplex (NC) formulation after various insults. Na, K-ATPase activity was measured by the spectrophotometric measurement of phosphate released from ATP with the use of ammonium molybdate. The Na, K-ATPase activity was calculated as the difference in ATPase activity between cells exposed to ouabain and those not exposed. Ussing chamber was used to measure the pump function of endothelial cells. Western blot analysis and immunocytochemistry were performed to measure the expression of Na, K-ATPase α₁ subunit.

Results : The Ca²⁺ NT NCs were prepared at a [Ca²⁺]:[NT]:[bPEI _1.8kDa] ratio of 0.25 mM: 0.5 mM: 1.0 mM and these preparations significantly and gradually increased the activity of Na, K-ATPase in cultured bovine corneal endothelial cells. And these effects were effectively blocked by the PKC inhibitor (protein kinase C). In addition, Ca²⁺ NT NCs (0.25 mM and 0.5 mM) increased the viability of cells exposed to a low temperature (4°C) for 4 or 8 hours in a serum containing medium (p<0.05). Western blot analysis indicated that Ca²⁺ NT NCs formulation significantly decreased the ratio of inactive Na, K-Atpase α₁ subunit (after 12 hours of incubation with 0.5 mM of Ca²⁺ NT NCs formation). Immunocytochemistry reveals that Ca²⁺ NT NCs formulation increased the cell surface expression of the Na,K-ATPase α₁ subunit.

Conclusions : This study demonstrates that the Ca²⁺ NT NC formulation increases the activity of Na, K-ATPase in the corneal endothelial cells to enhance the pump function. Our results imply a potential therapeutic strategy that the pumping function of the corneal endothelial cells can be improved by the administration of Ca²⁺ NT NC formulation, which may help to maintain and improve transparency of the cornea. Further experiments are to be carried out to confirm the practical effect of the Ca²⁺ NT NC formulation in vivo.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.