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HJ Gukasyan, VH L Lee, KJ Kim, R Kannan; Restoration by GSH and its Ester of Oxidant Induced Decrease in ISC Across Pigmented Rabbit Conjunctiva . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2465.
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Purpose: To test the hypothesis that oxidation can modify the thiol status of factors governing ion transport mechanisms (i.e. regulatory intermediates, pumps, or channels), the effect of hydrogen peroxide (H2O2) and (pre)treatment with GSH or its analog on the short circuit current (Isc comprised of active Na+-absorption and Cl--secretion) across the pigmented rabbit conjunctiva was studied. Methods: A modified Ussing chamber setup was used to examine the effect of H2O2 on conjunctival Isc across the freshly excised tissue. All experiments were performed in bicarbonated Ringer's solution at 37ºC under short-circuit conditions with the use of an automatic voltage clamp device. H2O2 was applied in the mucosal chamber at 0.05 to 50 mM to establish a dose response profile and to derive IC50. GSH (1-10 mM) and GSH monoethyl ester (GSH-MEE, 1-5 mM) were superfused over the mucosal surface of the conjunctiva. Total intracellular GSH levels were determined in H2O2-treated and untreated tissues. Results: Physiological concentrations of H2O2 (up to 100 µM) did not elicit any significant changes in Isc when compared to non-treated tissue. However, at ≷1 mM H2O2 a significant inhibition in Isc was observed (≷50% vs. controls, p<0.01). The IC50 for H2O2 was 1.44 0.18 mM. Superfusion with 10 mM GSH or with 5 mM GSH-MEE resulted in a significant recovery of Isc approaching values close to that of untreated controls (45 5%, p<0.01). Pretreatment (45 min) of conjunctivae with either GSH (5 mM) or GSH-MEE (2 mM) completely protected the tissues from H2O2-induced decrease in Isc. Conclusion: These data suggest an important role for GSH supplementation in oxidant-induced conjunctival injury. The restoration of Isc by GSH or GSH-MEE suggests that GSH regulates the thiol status of ion transporters or regulatory intermediates in conjunctival tissue, in addition to reduction of peroxides produced by oxidant insult.
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