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A Beyer-Mears, FP J Diecke; Effect of High Galactose Media on Intracellular Electrolyte and Myo-Inositol Concentrations in Human Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3541.
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Purpose: To determine in cells of a human lens cell line (HLE-3B) the changes in intracellular electrolyte concentrations and the cause of loss of myo-inositol (MI) subsequent to incubation in high galactose (GA). Methods: Cells were cultured to confluence and then exposed to media containing 30 mM GA for up to 4 days and intracellular electrolyte and MI concentrations were measured at different time points. Intracellular Na ([Na]i) and K ([K]i) concentrations were determined using atomic absorption spectroscopy (Perkin-Elmer Model 603) and chloride ([Cl]i) concentrations were measured by coulometric titration with a digital chloridometer (Labconco). MI concentrations and fluxes were determined with gas chromatography and labeled MI, respectively. K influx was determined using 86Rubidium. Results: Upon incubation in high GA medium there was an immediate increase in bumetanide- sensitive K influx due to cell shrinkage and activation of the Na-K-2Cl cotransporter. As a consequence [K]i, [Na]i and [Cl]i increased significantly compared to controls. This was followed by an increase in intracellular GA. The uptake of GA with obliged water resulted in volume increase which then activated a K-Cl cotransport reducing [K]i and [Cl]i. After 24 hours of incubation intracellular GA had equilibrated with the medium GA, while [K]i was decreased below control levels and [Cli to approximately control levels. [Na]i remained elevated thus reducing the Na gradient. During the following days the level of galactitol began to increase and this was accompanied by a further decrease in [K]i and [Cl]i. In parallel with the changes in intracellular electrolyte concentrations there was a decrease in intracellular MI which progressed from 18 mM in control cells to approximately 4 mM after 3 days in high galactose. This decrease was due a significant reduction of uptake and slight increase in efflux. Conclusions: Incubation in high GA media reduced the Na gradient and intracellular ionic strength. The reduced Na gradient lowers MI uptake and the reduced ionic strength increases MI efflux, thus leading to a reduction of intracellular MI.
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