The Effects of Elevated Glucose Upon Na+/K+-ATPase in Bovine Retinal Pigment Epithelial Cells

Crider, Julie Y., The Effects of Elevated Glucose Upon Na+/K+-ATPase in Bovine Retinal Pigment Epithelial Cells. Doctor of Philosophy (Biomedical Sciences, Pharmacology), December, 1994, 154 pp., 14 tables, 31 illustrations, bibliography, 288 titles. Bovine retinal pigment epithelial (RPE) cells were cultured under 1, 4.5 and 10 g/l glucose conditions in order to characterize the effects of hyperglycemia upon Na+/K+-ATPase. Functional activity of Na+/K+-ATPase was measured as ouabain-sensitive Rb+ uptake. 3H ouabain was used to assess binding characteristics of Na+/K+-ATPase. The major contributors to rubidium (mRb+) uptake activity were the ouabain-sensitive Na+/K+-ATpase and a bumetanide-sensitive NA+/K+/Cl- cotransporter. Dose response curves for ouabain and bumetanide produced IC50 values for 86Rb+ uptake of 60-100 nM and 120 nM, respectively. At elevated glucose concentrations, the aldose reductase inhibitor (ARI) AL-1576 stimulated 86Rb+ uptake upon chronic treatment. A sensitive new nonradioactive Rb+ uptake assay was developed which utilized suppressed conductivity detection and provided several advantages over the radioactive method. The average ouabain IC50 value was confirmed to be 100nM and was not significantly affected by elevated glucose concentrations. The bumetanide sensitive component was responsible for approximately 30% of Rb+ uptake at all glucose concentrations. Potassium efflux out of the cells was observed that was sensitive to the Na+/K+/Cl- cotransport inhibitor bumetanide. Elevated glucose appeared to increase Rb+ transport through potassium channels was also reduced Rb+ uptake indicating a decrease in Na+/K+-ATPase activity. Bovine RPE cells exposed to both high glucose and AL-1576 for one month showed mild stimulation of Rb+ uptake compared to the activity in high glucose alone. Ouabain and strophanthidin inhibition of 3H ouabain binding, in bovine RPE cells, appeared to be unaffected by hyperglycemia. The average IC50 values for these compounds were 5.02 x 10^-8 M, respectively. The results of this study indicate that Na+/K+-ATPase activity in bovine RPE is decreased by hyperglycemic state, and can be stimulated by treatment with an aldose reductase inhibitor administered from the onset of the hyperglycemic insult.