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VL Lee, HJ Gukasyan, R Kannan, KJ Kim; Mechanism of Net Glutathione (GSH) Secretion Into the Pigmented Rabbit Conjunctival Mucosa . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3260.
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Purpose: To characterize GSH transport mechanisms across primary cultured pigmented rabbit conjunctival epithelial cells (RCEC). Methods: RCEC were cultured on permeable support at an air interface. Kinetics of the effect of varying concentrations (5 - 135 mM) of sodium on GSH uptake from apical or basolateral fluids was performed using nonlinear least-squares regression computer analysis of data. The linear rates of GSH efflux from primary cultured RCEC were determined in cells maintained in NaCl-buffer (135 mM NaCl), choline chloride-buffer (135 mM choline-Cl-), high K+ buffer (135 mM KCl and 5 mM NaCl) with or without 1 µM potassium ionophore valinomycin, and NaCl-buffer with 1 µM valinomycin. Results: Hill coefficients for apical (1.2:1) and basolateral (1.3:1) GSH uptake as a function of [Na+] were not significantly different from unity, suggesting at least a 1:1 coupling ratio for Na+:GSH for the Na+-dependent uptake process. The Km in both apical and basolateral uptake studies for Na+ was ∼14 1 mM and Vmax of 29 2 pmol/106 cells for apical studies and 19 2 pmol/106 cells for basolateral uptake were found. The apical GSH efflux was slower in the presence than in the absence of Na+. There was no significant difference in GSH efflux for the two buffers into basolateral fluid with or without Na+. Depolarization of RCEC layers in apical high K+-buffer resulted in ∼90% reduction of GSH efflux to both apical and basolateral fluids. Valinomycin did not affect the high K+ effect. However, hyperpolarizing cell membranes with 1 µM valinomycin in the NaCl incubation buffer stimulated efflux rates more than two-fold into apical fluid. No detectable effect on basolateral efflux was observed. A net GSH secretion (∼1 nmol/cm2/hr) into apical fluid occurred under physiological conditions. Conclusion: Membrane potential-driven GSH efflux was a major component of GSH transport in conjunctival epithelial cells. The significance of net GSH secretion may lie in the protection of conjunctiva from chemical and oxidant insults.
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