Specialized protective role of mucosal glutathione in pigmented rabbit conjunctiva.
OBJECTIVE
To investigate mechanisms of H(2)O(2)-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 H(2)O(2) at which 50% of I(sc) was inhibited (IC(50)) were determined for the mucosal and serosal instillation of the agent. The rate of exogenous H(2)O(2) 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 H(2)O(2) at IC(50). Specific binding of (3)H-ouabain to the serosal side of the conjunctiva was estimated in the presence of mucosal or serosal H(2)O(2) to assess the role of functional Na(+)/K(+)-ATPase pumps in H(2)O(2) 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 H(2)O(2) decreased conjunctival I(sc) gradually in a dose-dependent manner. The mucosal IC(50) of H(2)O(2)was 1.49 +/- 0.20 mM, whereas the serosal IC(50) was estimated at 10.6 +/- 2.0 micro M. The rate of H(2)O(2) consumption from mucosal fluid was six times faster than that from serosal fluid. Conjunctival tissues pretreated with mucosal H(2)O(2) at IC(50) 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 H(2)O(2). When conjunctiva was bathed with Na(+)-free BRS on both the mucosal and serosal sides, before or after addition of mucosal H(2)O(2), 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 H(2)O(2) at IC(50) recovered to 60% to 80% of the initial pre-H(2)O(2) I(sc) after approximately 100 minutes. The specific binding of (3)H-ouabain to the serosal side of the tissue was inhibited by 85% in the presence of mucosal or serosal treatment with H(2)O(2) at their respective IC(50) 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 H(2)O(2) at IC(50). Furthermore, mucosal posttreatment with 10 mM GSH or 5 mM GSH-MEE of conjunctival tissues pre-exposed to mucosal H(2)O(2) resulted in a 30% recovery of the ouabain-binding level above that observed in tissues exposed to 1.5 mM H(2)O(2) alone on the mucosal side. By contrast, the decrease in conjunctival I(sc) or in the ouabain-binding level elicited by serosal H(2)O(2) at IC(50) was irreversible.


CONCLUSIONS
A higher mucosal IC(50) of [H(2)O(2)] on conjunctival I(sc) corresponds to the faster consumption of exogenous H(2)O(2) from mucosal bathing fluid. In addition, actively secreted GSH by conjunctival epithelial cells may help reduce the injury by mucosally applied H(2)O(2). Injury by H(2)O(2) 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.
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology; D002463:Cell Membrane Permeability; D003228:Conjunctiva / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019610:Cytoprotection; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005978:Glutathione / Q000494:pharmacology / Q000502:physiology*; D006861:Hydrogen Peroxide / Q000633:toxicity; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D008297:Male; D009092:Mucous Membrane / Q000187:drug effects / Q000378:metabolism; D010042:Ouabain / Q000378:metabolism; D016877:Oxidants / Q000633:toxicity; D018384:Oxidative Stress; D018408:Patch-Clamp Techniques; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D016877:Oxidants; D002034:Bumetanide; D015124:8-Bromo Cyclic Adenosine Monophosphate; D010042:Ouabain; D006861:Hydrogen Peroxide; D000254:Sodium-Potassium-Exchanging ATPase; D005978:Glutathione