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Hovhannes J. Gukasyan, Kwang-Jin Kim, Ram Kannan, Robert A. Farley, Vincent H. L. Lee; Specialized Protective Role of Mucosal Glutathione in Pigmented Rabbit Conjunctiva. Invest. Ophthalmol. Vis. Sci. 2003;44(10):4427-4438. doi: 10.1167/iovs.03-0437.
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purpose. To investigate mechanisms of H2O2-induced reduction in rates of active ion transport (Isc) across the pigmented rabbit conjunctival tissue and the protective role afforded by mucosal glutathione (GSH).
methods. Changes in Isc and specific binding properties of ouabain were evaluated in a modified Ussing chamber setup, using conjunctival tissues freshly excised from pigmented rabbits. Effective concentrations of H2O2 at which 50% of Isc was inhibited (IC50) were determined for the mucosal and serosal instillation of the agent. The rate of exogenous H2O2 consumption in the mucosal and serosal bathing fluids was estimated. Mucosal 8-Br cAMP at 3 mM, serosal bumetanide at 0.5 mM, and both mucosal and serosal bathing of the conjunctiva with Na+-free bicarbonated Ringer’s solution (BRS) were used to estimate contributions of conjunctival ion transport mechanisms in Isc changes elicited by mucosal H2O2 at IC50. Specific binding of 3H-ouabain to the serosal side of the conjunctiva was estimated in the presence of mucosal or serosal H2O2 to assess the role of functional Na+/K+-ATPase pumps in H2O2 injury. The effect of mucosally instilled GSH and other reductive and nonreductive agents on possible restoration of oxidant-induced decrease in conjunctival Isc was also determined.
results. Mucosal and serosal H2O2 decreased conjunctival Isc gradually in a dose-dependent manner. The mucosal IC50 of H2O2was 1.49 ± 0.20 mM, whereas the serosal IC50 was estimated at 10.6 ± 2.0 μM. The rate of H2O2 consumption from mucosal fluid was six times faster than that from serosal fluid. Conjunctival tissues pretreated with mucosal H2O2 at IC50 retained approximately 50% of their maximum 8-Br cAMP-dependent increases in Isc. Serosal bumetanide did not further reduce the Isc beyond the initial 70% decrease caused by mucosal H2O2. When conjunctiva was bathed with Na+-free BRS on both the mucosal and serosal sides, before or after addition of mucosal H2O2, the combined effects were additive, decreasing Isc by up to 95% to 99%. Mucosal, but not serosal, GSH or reduced l-glutathione mono-ethyl ester (GSH-MEE) superfusion of conjunctival tissues pre-exposed to mucosal H2O2 at IC50 recovered to 60% to 80% of the initial pre-H2O2 Isc after approximately 100 minutes. The specific binding of 3H-ouabain to the serosal side of the tissue was inhibited by 85% in the presence of mucosal or serosal treatment with H2O2 at their respective IC50 values. Pretreatment for 60 minutes with either 5 mM GSH, 2 mM GSH-MEE, or 0.1 mM ebselen, when instilled into the mucosal fluid, resulted in 30%, 45%, or 55% reductions, respectively, in ouabain binding after exposure to mucosal H2O2 at IC50. Furthermore, mucosal posttreatment with 10 mM GSH or 5 mM GSH-MEE of conjunctival tissues pre-exposed to mucosal H2O2 resulted in a 30% recovery of the ouabain-binding level above that observed in tissues exposed to 1.5 mM H2O2 alone on the mucosal side. By contrast, the decrease in conjunctival Isc or in the ouabain-binding level elicited by serosal H2O2 at IC50 was irreversible.
conclusions. A higher mucosal IC50 of [H2O2] on conjunctival Isc corresponds to the faster consumption of exogenous H2O2 from mucosal bathing fluid. In addition, actively secreted GSH by conjunctival epithelial cells may help reduce the injury by mucosally applied H2O2. Injury by H2O2 may directly affect vital membrane components (e.g., Na+,K+-ATPase) involved in active ion transport across conjunctiva. Mucosal protection by GSH (or its analogues) of active conjunctival ion transport may be useful in maintaining the physiological functions of conjunctiva under oxidative stress.
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