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S. Hato, H. Mochizuki, M. Yamada; The Effect of Dexamethasone on Pump Function and Na,K-ATPase Activity of Cultured Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2687.
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Recent studies have suggested that corticosteroids affect the pump function of corneal endothelium directly in addition to their anti-inflammatory effects. Pump function of corneal endothelial cells is mainly dependent on their Na,K-ATPase activity. This study was undertaken to test whether dexamethasone influences the pump function and Na,K-ATPase activity of mouse corneal endothelial cells.
C3H cell line derived from mouse corneal endothelial cells was used in the study. Various concentrations of dexamethasone were added to confluent C3H cells for 48 hours. C3H cells were permeabilized by osmotic shock and freeze-thaw, placed in a centrifuge, and the aliquot of the supernatant was equally divided in two wells. 10-5M ATP and 10-3M ouabain were added to one well, and only 10-5M ATP was added to another. The ATPase activity was estimated by phosphorus determination with ammonium molybdate, which was resulting from ATP hydrolysis. The Na,K-ATPase activity was calculated as the difference ATPase activity in the presence and absence of ouabain. The pump function of confluent monolayer of C3H cells was measured as short-circuit-current in an Ussing chamber. Confluent monolayer of C3H cells was mounted in the Ussing chamber and incubated in Kreb’s solution. After steady state level of the short-circuit-current was reached, 10-3M ouabain was added to the chamber, and short-circuit-current redetermined. The pump function was calculated as the difference short-circuit-current in before and after addition of ouabain.
Dexamethasone caused a dose-dependent activation of Na,K-ATPase at concentration from 10-8 M to 10-5M. The Na,K-ATPase activity reached 368% of control when 10-5M of dexamethasone was added. The endothelial pump function determined by an Ussing chamber system was also enhanced by dexamethasone in a dose dependent fashion, and reached 950% of control in the presence of 10-5M dexamethasone.
Our results indicated that dexamethasone increased the pump function of cultured corneal endothelial cells by activating Na,K-ATPase. Corticosteroids may be effective in treating corneal edema by activating the function of corneal endothelium.
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