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

AbstractPurposeNa, 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.MethodsBovine 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.ResultsThe Ca2+ NT NCs were prepared at a [Ca2+]:[NT]:[bPEI 1.8 kDa] 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.ConclusionsThis 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.