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Specialized Protective Role of Mucosal Glutathione in Pigmented Rabbit Conjunctiva

¹From the Departments of Pharmaceutical Sciences, ²Medicine, ⁷Ophthalmology, ³Physiology and Biophysics, ⁹Biochemistry and Molecular Biology, ⁴Biomedical Engineering, and ⁵Molecular Pharmacology and Toxicology; the ⁸Doheny Eye Institute; and the ⁶Will Rogers Institute Pulmonary Research Center, Schools of Pharmacy, Medicine, and Engineering, University of Southern California, Los Angeles, California.

PURPOSE. To investigate mechanisms of H₂O₂-induced reduction in rates of active ion transport (I_sc) across the pigmented rabbit conjunctival tissue and the protective role afforded by mucosal glutathione (GSH).

METHODS. Changes in I_sc 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 I_sc 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 I_sc 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 I_sc was also determined.

RESULTS. Mucosal and serosal H₂O₂ decreased conjunctival I_sc 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 I_sc. Serosal bumetanide did not further reduce the I_sc 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 I_sc 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 I_sc 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 I_sc or in the ouabain-binding level elicited by serosal H₂O₂ at IC₅₀ was irreversible.

CONCLUSIONS. A higher mucosal IC₅₀ of [H₂O₂] on conjunctival I_sc 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.