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H.J. Gukasyan, R. Kannan, K.J. Kim, R.A. Farley, V.H. Lee; Mucosal Glutathione Restores H2O2-Induced Decrease in Active Ion Transport Across the Pigmented Rabbit Conjunctiva . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3458.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate mechanisms of H2O2-induced reduction in active ion transport (Isc) across the pigmented rabbit conjunctival tissue and the protective role of mucosal glutathione (GSH). Methods: Changes in active ion transport (Isc) and the specific binding of ouabain were evaluated in bicarbonated Ringer’s solution (BRS), Na+-free BRS, or K+-free BRS at 37°C in a modified Ussing chamber setup using conjunctival tissues freshly excised from pigmented rabbits. Na+-free BRS and 10 µM serosal bumetanide were used to estimate the contribution of active Na+ and Cl- transport, respectively, to the changes in Isc induced by H2O2 (mucosal IC50, 1.44 mM; serosal IC50, 10.3 µM). Binding studies using serosal 15 nM 3H-ouabain in K+-free BRS were carried out to estimate the effect of H2O2 on the abundance of functional Na+/K+-ATPases. Finally, the effectiveness of added mucosal GSH in the protection of conjunctival tissues against oxidant-induced reduction in Isc was also studied. Results: Na+-free BRS and H2O2 induced sustained decrease in conjunctival Isc in an additive fashion (total 90-95% decrease). Bumetanide did not further decrease the 70% inhibition of Isc caused by H2O2. The specific binding of 3H-ouabain was inhibited by 85% in the presence of mucosal or serosal IC50 [H2O2]. When either 5 mM GSH, 2 mM GSH-monoethyl ester (GSH-MEE), or 0.1 mM ebselen was added to mucosal fluid for a 60 min pretreatment, 30, 45, and 55% reductions in ouabain binding were observed, respectively. Furthermore, post-treatment of conjunctival tissues previously exposed to H2O2 with mucosal 10 mM GSH or 5 mM GSH-MEE resulted in 25-30% recovery in the control physiological ouabain binding level. Conclusions: These data suggest that H2O2-induced decrease in active ion transport across conjunctival tissues more readily affects the Cl--dependent (but not Na+) component of Isc. Protection by GSH (or analogs) of functional Na+/K+-ATPases in the serosal membranes of conjunctiva may be useful in maintaining the physiological activity of conjunctiva under oxidative stress.
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