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(Investigative Ophthalmology and Visual Science. 2003;44:4427-4438.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.03-0437
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Specialized Protective Role of Mucosal Glutathione in Pigmented Rabbit Conjunctiva

Hovhannes J. Gukasyan,1 Kwang-Jin Kim,2,3,4,5,6 Ram Kannan,7,8 Robert A. Farley,3,9 and Vincent H. L. Lee1,7

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.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
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.

Glutathione (GSH) is the most prevalent, endogenous, thiol-containing tripeptide found in nearly all cell types, and constitutes more than 95% of total non-protein-associated cellular sulfur. Although additional physiological roles for GSH are rapidly emerging, one of its most commonly known functions is the protection of cells from a variety of oxidative insults. Glutathione has also been implicated in the direct modulation of several enzymes, where it is an essential cofactor. The thiol (-SH) status of cellular proteins is maintained by GSH by reducing disulfide bonds resulting from oxidant stress. Glutathione is biosynthesized within the cytoplasm in two steps, from the precursor amino acids L-glutamate, L-glycine, and L-cysteine. The availability of L-cysteine is the limiting factor in GSH biosynthesis.1 2 Various mechanisms that modulate cellular levels of GSH or L-cyst(e)ine have been shown to be closely regulated by oxidative means.3

Efflux of intracellular GSH occurs in its reduced or oxidized form, or as a chemical conjugate with toxins, predominantly contributing to the turnover of cellular GSH. Secretion of reduced GSH into the luminal fluid lining polarized epithelia of intestines, kidney, upper airway, and ocular surface has been shown.4 5 6 7 8 Using primary conjunctival epithelial cell layers from pigmented rabbits, we reported net secretion of intact GSH under physiological conditions.6 The conjunctival epithelium that covers most of the ocular surface is thought to function as a protective barrier and to participate in the maintenance of tear film stability by means of the mucus secreted from resident goblet cells.9 10 Potential damage emanating from light, heat, chemicals, atmospheric gases, or physical abrasion can be inflicted locally on the conjunctiva because of its immediate proximity to the surrounding environment.11 The thin tear film covering the conjunctiva serves as the front line of defense against environmental harm. A continuous supply of endogenous antioxidants is thought to be essential, considering that the tear film is a compartmentalized discrete milieu. The presence of several antioxidant molecules (e.g., cysteine, ascorbate, glutathione, urate, and tyrosine) in tear fluid secretions has been recognized. Their origin may be in the lacrimal gland and/or the conjunctiva.2

Conjunctival short-circuit current (Isc), measured under zero voltage-clamp conditions, essentially describes the algebraic sum of all active, energy-driven, and electrogenic ion transport processes occurring across the tissue. Dependency on temperature and the inhibitory effect of serosal ouabain (a cardiac glycoside that blocks the active efflux of Na+ and reuptake of K+, with characteristics of saturable and specific binding to serosal Na+,K+-ATPase pumps) on the rabbit conjunctival Isc are characteristic of active Na+,K+-ATPase-driven ion transport in this tissue.12 Immunocytochemical analysis of conjunctival tissues reveals an intense staining by anti-{alpha}1-subunit antibodies of Na+,K+-ATPase exclusively at the serosal surface.13 Inhibition studies of rabbit Na+,K+-ATPase indicate a model with two binding sites for ouabain: a high-affinity (Ki ~16 nM) and a low-affinity (Ki ~4.2 µM) site.14 Measurements of tissue Isc or whole cell membrane conductance and currents have been used as suitable endpoints in characterizing the effect of oxidative stress on ion transport across a number of epithelia. Related studies of Isc in rat tracheal15 or alveolar epithelial cell monolayers cultured on permeable support,16 as well as in several ocular tissues, including those of the amphibian corneal epithelium17 and the human fetal retinal pigment epithelium,18 19 have been reported. t-Butylhydroperoxide-induced changes in solute permeability and ion permeation selectivity in rat tracheal epithelium,15 as well as hydrogen peroxide (H2O2) and GSH modulation of specific Cl- channels expressed in the human retinal pigmented epithelium,20 have been considered in light of the importance of epithelial ion transport processes in the regulation of cellular pH and the volume or levels of extracellular fluid.21 22

To identify the role of conjunctival mucosal GSH in ocular surface health, we tested the hypothesis that exogenously instilled oxidants can directly alter conjunctival permeability to ions by evaluating the effects of H2O2 injury and GSH protection on the Isc of excised pigmented rabbit conjunctival tissues. We sought to determine whether mucosal supplementation with exogenous GSH can prevent or reverse injurious effects of H2O2 on active ion transport across conjunctival epithelium. We also studied the rate of consumption of exogenously added H2O2 in this model and evaluated whether treatments with H2O2 or GSH influences total conjunctival GSH level.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Animals and Reagents
The investigations using Dutch belted rabbits (pigmented males weighing 2.2 to 2.5 kg, Irish Farms, Los Angeles, CA) described in this report conform to the Guiding Principles in the Care and Use of Animals (Department of Health, Education and Welfare Publication, National Institutes of Health 80-23) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Hydrogen peroxide solution (H2O2, 30% wt/wt), reduced L-glutathione (g-glu-cys-gly; GSH), reduced L-glutathione mono-ethyl ester (g-glu-cys-gly-OEt; GSH-MEE), bumetanide (3-aminosulfonyl-5-butylamino-4-phenoxybenzoic acid), ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), DL-dithiothreitol ((±)-threo-1,4-dimercapto-2,3-butanediol; DTT), 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br cAMP), 2-mercaptoethanol, D-mannitol, sucrose, ouabain, and bovine serum albumin (96% pure; lyophilized powder) were all obtained from Sigma-Aldrich Co. (St. Louis, MO). [3H(G)]-ouabain (specific activity 18 Ci/mmol) was purchased from PerkinElmer Life Science Products, Inc. (Boston, MA).

Buffer Solutions
Experiments were conducted in normal, Na+-free, or K+-free bicarbonated Ringer’s solution (BRS) maintained at 37°C and pH 7.4 under 95% air/5% CO2. The normal BRS contained 110 mM NaCl, 5 mM KCl, 30 mM NaHCO3, 1.0 mM NaH2PO4, 1.0 mM CaCl2 · 2H2O, 0.75 mM MgCl2 · 6H2O, and 5 mM D-glucose. Na+-free BRS was prepared by equimolar replacement of NaCl, NaH2PO4, and NaHCO3 with choline chloride, KH2PO4, and choline bicarbonate, respectively. K+-free BRS was prepared by equimolar replacement of KCl with NaCl. The osmolality of normal, Na+-free, and K+-free BRS was adjusted to 300 mOsm/kg H2O with sucrose, as needed.

Short-Circuit Current Recording
Pentobarbital sodium solution at 85 mg/kg was administered via a marginal ear vein to kill the rabbits, and the entire eyeball was surgically removed from the orbit to excise conjunctival epithelium from adjoining ocular tissues. The excised conjunctiva was mounted in a special tissue adapter with a circular aperture of 0.96 cm2. The adapter-tissue assembly was installed in a modified Ussing chamber and maintained at 37°C by a circulating water bath. The bathing solution (6 mL on each side) was bubbled with 95% air/5% CO2 to maintain pH at 7.4 and to provide sufficient mixing of the solution.12

Short-circuit conditions were achieved with a automatic voltage-clamp device (model 558C-5; Bioengineering Department, University of Iowa, Iowa City, IA), which was externally controlled by custom-programed computer software (SP System 2002) with methods for external command of the voltage clamp device (558C-5). The software is a LabView (National Instruments [NI], Austin, TX) graphical G-programming-language-based program used for analog device control and simultaneous digital data acquisition, analysis, and presentation. The system comprises a data acquisition board (NI-DAQ model PCI 6024E), linked to an external analog-to-digital converter interface box (NI-BNC 2120), which was connected to the voltage-clamp device with cables (Bayonet Navy Connector cables; BNC) (minimum computing requirements for LabView, NI-DAQ, and NI-BNC 2120 can be found at http://www.ni.com/ National Instruments Corp.). The potential difference across conjunctival tissues was measured with a pair of calomel electrodes, and the direct current flowing across the tissue was transmitted with a pair of Ag/AgCl electrodes. The Isc (in microamps per square centimeter) was monitored and digitally recorded with the SP System 2002. A separate module of SP System 2002, "Resistance," was used to estimate and automatically compute the transepithelial electrical resistance (TEER) of tissues. Briefly, the Resistance module was programed to impose a 2-mV pulse for 3 seconds at 1-minute intervals across the short-circuited tissue to calculate the TEER using Ohm’s law as a surface-area-normalized ratio of applied voltage pulse to the resultant current.12 23 24 Measurements of all non-Isc endpoints (e.g., H2O2 levels in bathing fluids, total tissue GSH level, and 3H-ouabain binding) described in the following sections were performed when Isc reached the steady state level after each treatment.

Conjunctival Studies of Isc on H2O2 Dose-Response and Treatment with Other Reagents
The effect of hydrogen peroxide on conjunctival Isc was studied as a function of increasing concentrations applied to either mucosal or serosal normal BRS bathing the conjunctival tissue at 37°C. Hydrogen peroxide-induced changes in conjunctival Isc were expressed as a percentage of initial Isc, plotted against log[H2O2] in the bathing fluids, and analyzed with a sigmoidal dose-response plot with a variable slope. Kinetic parameters (e.g., IC50 and maximum inhibition) for the H2O2 dose-response in the conjunctiva were estimated by nonlinear least-squares regression analysis of the data on computer (Prism, ver. 3.0; GraphPad Software, San Diego, CA) and the equation

where X is the commercial logarithm of [H2O2], Y is the Isc, and bottom and top are Isc, where baseline value and maximum H2O2 effect are attained, respectively.

To determine ion transport mechanisms that may be influenced during inhibition of conjunctival Isc with mucosal (or serosal) H2O2 at IC50, conjunctival tissues were treated sequentially with H2O2 followed by a maneuver that is known to modulate Isc in the following combinations: (1) 3 mM mucosal 8-Br cAMP before or after mucosal 1.5 mM H2O2 in normal BRS, (2) 0.5 mM serosal bumetanide before or after mucosal 1.5 mM H2O2 in normal BRS, (3) Na+-free BRS superfusion of both mucosal and serosal sides before or after mucosal 1.5 mM H2O2 in normal or Na+-free BRS, respectively. The rationale for reversing the order of administering H2O2 and these agents was to determine the effect of H2O2-induced oxidant stress on normal conjunctival Isc, or the remaining Isc after the specific maneuver, which partially alters a component thereof. To determine whether the H2O2-induced decreases in conjunctival Isc could be restored with GSH treatment, all tissues were treated in normal BRS sequentially with mucosal or serosal H2O2 at IC50 and then with mucosal or serosal GSH (or a control reagent replacing GSH) and in the reverse order, for Isc measurements. Periods of exposure to each agent were determined by the amount of time it took for a given treatment to achieve a maximum effect on conjunctival Isc.

Estimation of Consumption Rates of Exogenous H2O2 Instilled into Normal BRS Bathing Fluids
Detection of H2O2 was performed with a colorimetric kit (Molecular Probes, Inc., Eugene, OR). Exogenous H2O2 at 100 µM was added simultaneously to the mucosal and serosal normal BRS bathing the conjunctival tissue mounted in the Ussing chamber. A control study was run simultaneously under identical conditions, replacing the conjunctival tissue with a parafilm to assess the rate of H2O2 autodegradation in normal BRS. An aliquot (50 µL) was taken from normal BRS control, mucosal, and serosal fluids bathing conjunctival tissues at predetermined time points (1, 3, 5, 10, 15, 20, and 30 minutes) and assayed for H2O2 activity. The detection assay uses a one-step 1:1 stoichiometry reaction between a red fluorescent label (Amplex Red; 10-acetyl-3,7-dihydroxyphenoxazine; Molecular Probes) and H2O2 to produce a red fluorescent oxidation product, resorufin (lmax, 563 nm). Samples and standards were loaded in a 96-well polystyrene flat-bottomed plate (EIA/RIA Plate; Corning-Costar, Cambridge, MA) and the amount of H2O2 was determined by measuring absorbance at 560 nm in a microplate reader (MRX Microplate Reader; Dynatech Laboratories, Chantilly, VA). First-order rate constants and half-lives (t1/2) of H2O2 were determined from the semilog plot of the integrated first-order velocity equation. All H2O2 consumption data were adjusted for autodegradation of H2O2 in parafilm experiments.

The first-order rate constant and half-lives for H2O2 consumption in the mucosal and serosal fluids bathing excised conjunctivas were estimated using the equation

where [S] represents the concentration of H2O2 at various time points t, k denotes the reaction (i.e., decomposition) rate, and S0 is the initial concentration of H2O2. Thus, a plot of log[H2O2] versus t is linear, with a slope of -k/2.3. When 50% of [H2O2]0 remains in the bathing fluid, the corresponding t is the t1/2, and the reaction rate k can be estimated from the equation 0.693/k = t1/2.

Estimation of Total Tissue GSH Level
Hydrogen peroxide at a final concentration of 1.5 mM was directly instilled into the normal BRS bathing the mucosal side of conjunctival tissues mounted in the modified Ussing chamber, while Isc was continually monitored on the SP System 2002. For serosal treatment, 15 µM H2O2 was used instead. These concentrations of H2O2 were chosen based on H2O2 dose-response studies of Isc (as described earlier). After the H2O2 effect reached steady state Isc, the tissues were mucosally superfused (posttreated) with normal BRS containing 10 mM GSH, 5 mM GSH-MEE, 5 mM DTT, 5 mM 2-mercaptoethanol, 10 mM D-mannitol, 10 mM sucrose, or 0.1 mM ebselen and allowed to incubate with these reagents in the modified Ussing chamber for approximately an additional 100 minutes.

Mucosal pretreatments of conjunctival tissues before mucosal instillation of H2O2 were also performed, where GSH and GSH-MEE were instilled at 5 and 2 mM, respectively, whereas all other reagents were used at the same concentration as used for posttreatment experiments. Total tissue GSH level was estimated by a colorimetric microplate assay kit (model GT20; Oxford Biomedical Research, Oxford, MI), before and after the treatments just described. The molecular form of GSH and other thiols (e.g., oxidized glutathione [GSSG], cysteine, and cystine) in isolated conjunctival tissue homogenates (prepared on ice using a Pyrex tissue grinder; 16 x 150 mm; Corning, Inc., Corning, NY) was verified according to an established chromatographic method.25

3H-ouabain-Binding Studies
To establish that the alterations in conjunctival Isc were associated with actively driven ion transport, labeled ouabain binding to the conjunctival serosal Na+,K+-ATPase was estimated in K+-free BRS. Potassium competes with ouabain for binding sites on functional Na+,K+-ATPase pumps, therefore KCl was replaced from the incubation buffer with equimolar NaCl.26 The procedure of Horowitz et al.26 was modified to determine levels of specific 3H-ouabain binding in conjunctival tissues. The concentration of serosal ouabain at which 50% of rabbit conjunctival tissue Isc is inhibited was reported by our laboratories to be approximately 20 µM with a t1/2 for Isc decrease of 41.5 ± 6.06 minutes. Preliminary studies established that steady state binding of serosal 3H-ouabain at 15 nM (without serosal K+) was reached after 80 minutes of incubation, and further incubation for a total of 180 minutes did not alter the overall amount of radioactivity associated with conjunctival tissues from the amount observed at 80 minutes. The Isc was measured during specific segments of 3H-ouabain binding studies to determine whether the maximum effect of (1) mucosal or serosal H2O2 at IC50, (2) mucosal 0.1 mM ebselen pretreatment, (3) mucosal 5 mM pre- or 10 mM posttreatment using GSH, and (4) mucosal 2 mM pre- or 5 mM posttreatment using GSH-MEE was achieved. Binding studies were initiated after the stable Isc from all treatments by adding 3H-ouabain at a final concentration of 15 nM to the K+-free BRS bathing the serosal side of the conjunctiva mounted in the modified Ussing chamber, while the mucosal side was bathed with the normal BRS. Tissues were incubated with 3H-ouabain for 80 minutes, followed by excision of the conjunctivas and washing them three consecutive times in 100 mL each of 1.5 mM unlabeled ouabain in ice cold K+-free BRS. The rationale for using unlabeled ouabain in the washing solution was to displace nonspecific association of the labeled form from conjunctivas, because the dissociation of specifically bound 3H-ouabain is slow at 4°C.26 After they were washed, the conjunctival tissues were placed in 1 mL of tissue-solubilizing solution (0.5 N NaOH and 15% Triton X-100 in K+-free BRS) for 1 hour at room temperature with continuous ultrasonication (185 W). Twenty microliters from each tissue homogenate was taken for a protein assay (DC; Bio-Rad, Hercules, CA) with bovine serum albumin as a standard, and the remainder was mixed with a liquid scintillation cocktail (Econosafe; Research Products International, Mount Prospect, IL) for a 3H-activity assay. The amount of 3H-ouabain associated with conjunctival tissue was determined by measuring 3H-activity in a liquid scintillation counter (LS1801; Beckman Instruments, Fullerton, CA). Nonspecific binding was determined by estimating 3H-ouabain binding as described earlier with the concomitant presence of 1.5 mM unlabeled ouabain during the 80-minute incubation period.

Statistical Analysis
All data are expressed as the mean ± SEM for 3 to 10 determinations. The unpaired, two-tailed Student’s t-test was used to determine the statistical difference between two group means, where applicable. Comparisons among three or more group means were performed by one-way analysis of variance (ANOVA). Statistical significance among the group (>=3) means was determined by the modified Fisher’s least-squares difference approach. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Effect of Mucosal or Serosal H2O2 on Conjunctival Isc
Figure 1 shows the decreases in Isc as a percentage of its value at t = 0 in response to increasing doses of H2O2. At physiological concentrations of H2O2 found within cells27 28 or in other avascular intraocular compartments2 (0–100 µM), Isc did not change significantly by mucosal application of H2O2. Hydrogen peroxide afforded approximately two orders of magnitude difference in 50% inhibition of conjunctival Isc from the mucosal versus serosal side. At concentrations of H2O2 that elicited significant decreases in Isc, steady state levels were reached within 15 minutes of H2O2 application.



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  		FIGURE 1. Dose-response of conjunctival Isc to various mucosal and serosal concentrations of H2O2 in normal BRS. The data show the percentage decreases in Isc observed at 15 minutes after H2O2 instillation. The IC50 for mucosal H2O2 was 1.49 ± 0.2 mM, and the IC50 for serosal H2O2 was 10.6 ± 2.0 µM. In the range of reported physiological concentrations (~1–50 µM) of H2O2, Isc was unaffected. The serosal aspect of excised conjunctiva was more sensitive by two orders of magnitude to the H2O2-induced decrease in Isc. The control Isc was approximately 10 µA/cm2. Data are the mean ± SEM of results in three to four tissues.

 
Effect of 8-Br cAMP, Bumetanide, and Na+-free BRS on Mucosal H2O2-Induced Isc Inhibition
A set of conjunctival tissues pretreated with mucosal (8-Br cAMP), serosal (bumetanide), or both fluids (Na+-free BRS), were posttreated with 1.5 mM H2O2 applied to mucosal fluid at the time of maximum effect induced by each of the three maneuvers. A second set of conjunctival tissues were treated identically, only the application of H2O2 and the three maneuvers were performed in the reverse order. Because serosal H2O2 caused irreversible and severe changes in conjunctival Isc, the following results describe only those studies performed in combination with mucosal H2O2 at IC50.

When conjunctival tissues were treated with mucosal 3 mM 8-Br cAMP, the conjunctival Isc increased above baseline by 66%. Mucosally applied H2O2 at maximum 8-Br cAMP effect drastically inhibited the Isc to 15% below the initial baseline (Fig. 2A) . For the reverse treatment sequence, Isc was inhibited by nearly 60% with 1.5 mM mucosal H2O2, which was elevated by posttreatment with mucosal 8-Br cAMP to a level of 36% above the H2O2-pretreated baseline.



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  		FIGURE 2. Time course of Isc changes in conjunctival tissues exposed to three specific manipulations each in combination with mucosal H2O2 (at IC50) pre- and posttreatment. (A) 8-Br cAMP-dependent stimulation of conjunctival Isc before and after mucosal H2O2. (B) Effects of serosal bumetanide (BUM) and (C) Na+-free conditions (in both the mucosal and serosal sides) on conjunctival Isc inhibition by mucosal H2O2. Arrows: times of application of 1.5 mM mucosal H2O2, 3 mM mucosal 8-Br cAMP, 0.5 mM serosal bumetanide, or superfusion of both the mucosal and serosal chambers with 60 mL Na+-free BRS. The 8-Br cAMP-dependent pathway of conjunctival Isc stimulation was inhibited 50% by mucosal H2O2 (A), but bumetanide did not affect it further (B) in normal BRS. Incubation of tissues in Na+-free BRS on both sides produced an additive inhibition of Isc with mucosal H2O2 (C). The superscripts (1st) and (2nd) designate the sequence with which respective treatments in each panel were introduced. Error bars are omitted from selected data points for clarity. Data are the mean ± SEM of results in three to four tissues.

 
Conjunctival tissues, when treated with 0.5 mM serosal bumetanide displayed a decrease in Isc of nearly 70%. At maximum bumetanide effect, mucosal application of 1.5 mM H2O2 did not cause a further decline in Isc. Conversely, when H2O2 was applied first, a 60% Isc decrease occurred, and additional treatment with bumetanide did not elicit further Isc inhibition (Fig. 2B) . When Na+-free BRS was used to bathe both mucosal and serosal sides of conjunctival tissues, Isc dropped by 30% below baseline. At maximum Na+-free effect, mucosally instilled 1.5 mM H2O2 elicited a further decrease in Isc to levels to near zero. For the reverse sequence of treatments, the initial 60% decrease in Isc caused by mucosal H2O2 was augmented by an additional decrease when both sides of conjunctival tissues were bathed with Na+-free BRS, leading to an Isc level that was not different from zero (Fig. 2C) .

Effect of GSH Treatment on H2O2-Induced Isc Impairment
The Isc inhibitory effect of serosal H2O2 at IC50 could not be protected or recovered using mucosal or serosal GSH, or any of the other combinations of treatments tested. By contrast, mucosally applied GSH protected Isc from 1.5 mM mucosal H2O2-induced conjunctival Isc decrease. When used at 1 and 2.5 mM, GSH was ineffective during mucosal pre- or posttreatments in recovery of Isc within the time course tested, whereas at 5 mM it was ineffective in Isc protection when used in posttreatments, but effective in pretreatments.

Pretreatment.
In pretreatment of conjunctival tissues with 5 mM GSH or 2 mM GSH-MEE in normal BRS in the mucosal bathing fluid for 60 minutes, Isc or TEER did not change significantly from baseline. In contrast, 0.1 mM ebselen pretreatment led to decreases in Isc and TEER by 45% and 85%, respectively (Table 1) . When these pretreated tissues were superfused from both the mucosal and serosal sides using 60 mL of normal BRS, the subsequent addition of 1.5 mM mucosal H2O2 did not elicit a change in Isc. The final Isc recorded at 60 minutes after mucosal H2O2 insult in these tissues was significantly higher than that in conjunctivas with mucosal H2O2 treatment alone (Table 1) . Pretreatments with GSH and GSH-MEE preserved the TEER of conjunctival tissues. However, mucosal pretreatment with ebselen did not protect TEER (Table 1) . When 15 µM serosal H2O2 was used on GSH-, GSH-MEE-, or ebselen-pretreated tissues, it still elicited a 60% decrease in Isc after 15 minutes of H2O2 treatment. Furthermore, when conjunctival tissues were pretreated with 10 mM D-mannitol, 5 mM DTT, 5 mM 2-mercaptoethanol, or 10 mM sucrose, subsequent application of mucosal or serosal H2O2 at IC50 still produced a 60% decrease in Isc.


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  		TABLE 1. Effect of Mucosal Pretreatment on Changes in Conjunctival ISC by Mucosally Applied H2O2

 
Posttreatment.
Exposure of conjunctival tissues to either 1.5 mM mucosal or 15 µM serosal H2O2 in normal BRS resulted in a consistent 60% to 70% reduction of Isc after 15 minutes of treatment (Fig. 3) . The decrease in Isc was accompanied with an 80% decrease in TEER (Table 2) . When conjunctivas were superfused with 60 mL of normal BRS after the effect of mucosal or serosal H2O2 reached maximum, the decrease in Isc (or that in TEER) brought about by mucosal or serosal H2O2 was not recovered (Table 2 , Fig. 3 ). When conjunctival tissues pre-exposed to 1.5 mM mucosal H2O2 were posttreated with mucosal 10 mM GSH, 5 mM GSH-MEE, or 10 mM D-mannitol, significant recoveries of Isc (by 60%, 80%, and 35%, respectively) and TEER (by 40%, 50%, and 40%, respectively) were observed compared with superfusion with normal BRS alone (Table 2) . Posttreatment with mucosal GSH-MEE at 5 mM resulted in the highest recovery of Isc and TEER, increasing by 80% and 50%, respectively, after 100 minutes (Table 2 , Fig. 3 ). When 15 µM serosal H2O2 was initially used, posttreatment with GSH, GSH-MEE, or D-mannitol did not lead to recovery of Isc or TEER. In paired treatments of conjunctivas using mucosal or serosal H2O2, followed by mucosal or serosal posttreatment with 5 mM DTT, 5 mM 2-mercaptoethanol, 10 mM sucrose, and 0.1 mM ebselen all failed to elicit improvements in Isc or TEER (Table 2) .



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  		FIGURE 3. Time course of Isc changes in conjunctival tissues pretreated with mucosal H2O2 at IC50, followed by posttreatments with mucosal GSH or GSH-MEE in normal BRS. Arrow: time of mucosal instillation of H2O2. Bracket: length of time of subsequent posttreatments (superfusions) with mucosal 10 mM GSH or 5 mM GSH-MEE in normal BRS or mucosal and serosal superfusion with fresh normal BRS. Error bars are omitted from selected data points for clarity. Data are the mean ± SEM of results in three to four tissues.

 

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  		TABLE 2. Effect of Mucosal Posttreatments on Changes in Conjunctival ISC by Mucosally Applied H2O2

 
All mucosal or serosal treatments were essentially ineffective in reversing the insult of serosal H2O2 at IC50 on conjunctival Isc or TEER. Furthermore, restoration of Isc from mucosal insult by H2O2 at IC50 was observed only when GSH (and other reagents) was administered from the mucosal side of conjunctival tissues. In the cases of mucosal GSH, GSH-MEE, and D-mannitol, the TEER of conjunctival tissues exposed to mucosal H2O2 at IC50 was relatively preserved over the entire 120 minutes of the experiments. Ebselen protected Isc in pretreatments, but not TEER, in that it did not rescue conjunctival tissue TEER in both pre- and posttreatments.

Estimation of Consumption Rates of Exogenously Instilled H2O2
H2O2 autodegraded with a first-order reaction rate constant (k) of 2.99 x 10-3/min in normal BRS in the absence of conjunctival tissues. The adjusted first-order rate constant for the disappearance of H2O2 from the mucosal bathing fluid of conjunctival tissues was 2.74 x 10-2/min, when an initial concentration of 100 mM H2O2 was used. In contrast, at the same starting concentration of H2O2 the first-order rate constant for the disappearance of H2O2 from the serosal fluid was 4.83 x 10-3/min (Fig. 4) . Thus, the consumption rate of exogenous H2O2 when applied to mucosal bathing fluid of conjunctivas appeared to be approximately six times faster than when added to the serosal bathing fluid.



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  		FIGURE 4. Detection of exogenously instilled H2O2 in normal BRS bathing the mucosal and serosal sides of conjunctival tissues mounted in a modified Ussing chamber at various times points. Data are shown as the remaining H2O2 level versus time. Consumption of H2O2 was determined from these curves after adjusting for control H2O2 autodegradation. The slopes of -13.2 x 10-3 (r2 = 0.97, {blacktriangleup}) and -3.4 x 10-3 (r2 = 0.96, {square}) for H2O2 consumption from mucosal and serosal fluids of normal BRS, respectively, were estimated. A t1/2 for H2O2 of approximately 25 and 143 minutes in respective bathing fluids of the conjunctiva was estimated using the relation, t1/2 = 0.693/(-2.3 x slope). Data are the mean ± SEM of results in three to four tissues.

 
Changes in Total GSH Level with H2O2 Treatment
The estimated amount of total GSH in homogenates of the freshly excised and Ussing chamber-mounted conjunctival tissues were in close agreement with our previous reports,6 ranging between 25 and 30 nmol/mg protein. Serosal H2O2 at IC50 did not cause a significant change in total GSH level after a 120-minute incubation. By contrast, a 35% to 40% decrease in conjunctival GSH was observed after incubation with 1.5 mM mucosal H2O2 for 120 minutes (Fig. 5) . Five mM dithiothreitol, 5 mM 2-mercaptoethanol, 10 mM sucrose, or 0.1 mM ebselen posttreatments failed to change the tissue GSH levels (data not shown). When tissues pretreated with mucosal H2O2 at IC50 were posttreated with 5 mM GSH-MEE for 100 minutes, a significantly higher level of total GSH was found than in the untreated control (Fig. 5) . Mucosal posttreatment with 10 mM D-mannitol after maximum H2O2 effect was reached led to recovery of Isc by 35% (Table 2) without any effect on total conjunctival GSH level (Fig. 5) . A similar 35% to 40% decrease in total conjunctival GSH occurred when conjunctival tissues pretreated with mucosal 10 mM D-mannitol, 10 mM sucrose, 5 mM DTT, or 5 mM 2-mercaptoethanol were subsequently exposed to mucosal H2O2 at IC50 (data not shown). Moreover, posttreatments with mucosal H2O2 at IC50 did not have an effect on total conjunctival GSH in the case of mucosal pretreatments with 5 mM GSH, 2 mM GSH-MEE, and 0.1 mM ebselen for 60 minutes (Fig. 5) .



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  		FIGURE 5. Changes in total cellular GSH level in excised conjunctiva were assayed after the maximum effect on Isc. Mucosal H2O2 at 1.5 mM caused a 40% decrease in GSH level (Mucosal IC50 [H2O2]), whereas serosal treatment with 15 µM H2O2 did not change the GSH level (Serosal IC50 [H2O2]). Posttreatment with mucosal mannitol at 10 mM did not change the total conjunctival tissue GSH level ((1st)H2O2, (2nd)mannitol). When mucosal GSH at 10 mM ((1st)H2O2, (2nd)GSH) and GSH-MEE at 5 mM ((1st)H2O2, (2nd)GSH-MEE) were used in posttreatments they significantly increased total conjunctival tissue GSH level compared to the mucosal IC50 [H2O2] condition. Total GSH levels did not change when a 60-minute pretreatments with 5 mM GSH ((1st)GSH, (2nd)H2O2), 2 mM GSH-MEE ((1st)GSH-MEE, (2nd)H2O2), and 0.1 mM ebselen ((1st)ebselen, (2nd)H2O2) were followed with mucosal H2O2 at IC50. Results are presented as the percentage of intracellular GSH level in treated conjunctival tissues compared with that of untreated tissues which were taken as 100%. {dagger}P < 0.05 vs. BRS control. {ddagger}Significant difference at P < 0.05 compared with total conjunctival tissue GSH level in response to mucosal H2O2 at IC50 (Mucosal IC50 [H2O2]). Data are the mean ± SEM of results in three to four tissues.

 
Changes in the Level of Ouabain Binding after H2O2 Treatment
Conjunctivas were treated with mucosal or serosal H2O2 at IC50 first, followed by mucosal or serosal application of GSH (or a particular control reagent replacing GSH) for estimation of the specific 3H-oubain binding to serosal membranes of tissues. A reverse sequence of application of agents used in the experiments was also performed. The level of serosal 3H-ouabain binding was 125.9 ± 21.5 fmol/mg protein for control conjunctivas (Fig. 6) . Nonspecific binding of 3H-ouabain was 6% to 8% (Fig. 6) . When 5 mM GSH, 2 mM GSH-MEE, or 0.1 mM ebselen was added to mucosal fluid for a 60-minute pretreatment, only 30%, 45%, and 55% reductions in ouabain binding were observed, respectively, after posttreatment with mucosal H2O2 at IC50. Consistent with the Isc studies, serosal H2O2 at IC50 also caused an 80% to 85% decrease in specific 3H-ouabain binding for these pretreated tissues. Posttreatments with mucosal 10 mM GSH or 5 mM GSH-MEE were ineffective in augmenting specific 3H-ouabain binding, when treated serosally after mucosal application of H2O2, or treated either mucosally or serosally after serosal H2O2 insult.



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  		FIGURE 6. Changes in specific 3H-ouabain binding in conjunctival tissues exposed to various mucosal and serosal treatments. When H2O2 was administered at 1.5 mM from mucosal fluid or 15 µM from serosal fluid (respective IC50s for Isc) it caused an 85% decrease in specific 3H-ouabain-binding mucosal IC50 [H2O2] and serosal IC50 [H2O2]), respectively. Pretreatments of conjunctival tissues for 60 minutes with mucosal 0.1 mM ebselen ((1st)ebselen, (2nd)H2O2), 5 mM GSH ((1st)GSH, (2nd)H2O2), and 2 mM GSH-MEE ((1st)GSH-MEE, (2nd)H2O2) all reduced the degree of inhibition in specific 3H-ouabain binding after 1.5 mM mucosal H2O2 (not with 15 µM serosal H2O2). Posttreatment of conjunctival tissues showing maximum mucosal H2O2 effect (at IC50) on Isc for approximately 100 minutes with 10 mM mucosal GSH ((1st)H2O2, (2nd)GSH) and 5 mM mucosal GSH-MEE ((1st)H2O2, (2nd)GSH-MEE) both recovered the level of specific 3H-ouabain binding by approximately 30% (not after 15 mM serosal H2O2). All data sets are illustrated with stacked bar graphs to compare the contribution of specific ({square}) and nonspecific ({blacksquare}) 3H-ouabain binding level with the total across all categories. {dagger}P < 0.05 versus control. {ddagger}Significant difference at P < 0.05 compared with specific 3H-ouabain binding in response to mucosal H2O2 at IC50 and versus control. Data are the mean ± SEM of results in three to four tissues.

 

   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
In this study, the effect of H2O2 on Isc flowing across freshly isolated pigmented rabbit conjunctival tissues was two orders of magnitude greater with serosal than mucosal application. Although specific electrophysiological changes observed in conjunctival tissues exposed to H2O2 were accompanied by a parallel decrease in total GSH, the basal tissue GSH levels did not change and in some cases even slightly increased when mucosal GSH or GSH-MEE was used in pre- or posttreatments. Variations in the specific 3H-oubain association with serosal membranes of tissues as a marker for functional Na+,K+-ATPase pumps suggested possible mechanisms of H2O2-induced decrease in conjunctival active ion transport, as well as for the protective role of mucosal (but not serosal) application of GSH. Oxidation induced changes in conjunctival ion transport suggest a critical role of conjunctival GSH metabolism and transport in ocular surface health.

Asymmetrical Effect of H2O2 on Isc
Transport characteristics of GSH in conjunctival epithelial cells under normal conditions, together with the mucosal presence of tear fluid, may be responsible for the observed more than a 100-fold difference in H2O2 sensitivity of conjunctival Isc in the serosal versus mucosal surface (Fig. 1) . Results of H2O2 treatment studies of active ion transport and resistance to passive solute flow were similarly asymmetric in rat alveolar epithelial cell monolayers cultured on permeable supports.16 H2O2 decreased alveolar epithelial Isc gradually in a dose-dependent manner from both apical and basolateral fluids, and the basolateral treatment was approximately 100 times more potent in IC50 concentration of H2O2. Furthermore, the sensitivity of Isc to apical H2O2 was inversely dependent on catalase (a ubiquitous heme protein that catalyzes the dismutation of H2O2 into water and molecular oxygen) activity, whereas that of the basolateral treatment was not.16 The activity of catalase is considered as important as GSH in cellular defense against H2O229 and has also been biochemically assessed within the lacrimal fluid of healthy and injured eyes.30 Minimally stimulated tear samples collected from human subjects do not display a detectable catalase or GSH-peroxidase activity, suggesting that the tear film may lack a GSH/GSSG cycle that provides significant protection from oxidative properties of H2O2 inside the cells.31

Amounts of H2O2 have been determined by chemiluminescent, radioisotopic, and enzymatic methods in the aqueous humor32 and lenses33 of several animal species, including humans. Estimated concentrations of H2O2 present in the aqueous humor of most species range from 5 to 41 µM, whereas in humans with cataracts the average level is 189 ± 88 µM in aqueous humor and approximately 100 µM in lens tissue.32 33 Furthermore, the levels of H2O2 have been shown to increase up to 20-fold in certain age-related diseases of the eye with oxidative stress characteristics.2 Because H2O2 is an ultimate byproduct of cellular respiration, it may be present in tear fluid secretions under normal conditions. It can also arise in vitro spontaneously from ascorbic acid (found in tear fluid at concentrations of 0.8–0.9 mM34 ), through a reaction with riboflavin (detected in ocular surface tissues of the rabbit35 ), and light or trace amounts of unbound metals. There may be a dynamic equilibrium for H2O2 through its continuous production in the ocular surface and tear film and subsequent elimination.2 In this regard, net GSH secretion by conjunctival epithelial cells may be a critical factor for the maintenance of this equilibrium.6 Considering a steady state GSH concentration to be 110 and 15 µM for the tear side and serosal side of the conjunctiva, respectively, the estimated intracellular GSH level is approximately 1 mM.6 The rate of elimination of exogenous H2O2 from normal BRS bathing the mucosal surface of conjunctival tissues in this study was six times faster than the rate measured at the serosal aspect. It has been reported that, in adult pigmented rabbits, buthionine sulfoximine (BSO, an inhibitor of GSH biosynthesis) increased the t1/2 of elimination of intracamerally injected 10 mM H2O2 by 77%, whereas suppression of catalase activity with 3-aminotriazole increased it by only 40%,36 indicating the dual involvement of GSH and catalase activity in elimination of H2O2 exogenously delivered to the eye. Whether the mucosal surface of the conjunctival epithelium exhibits similar catalase activity (either from tear source or conjunctival elaboration) is unknown.

Mechanisms of H2O2- and GSH-Induced Changes in Isc
Conjunctival epithelial cells display an active mucosal secretion of Cl-, a phenomenon estimated to account for approximately 70% of Isc of this tissue.12 Net conjunctival Cl- flux is modulated by at least three different mechanisms—cAMP, protein kinase C, and Ca2-mediated processes—suggesting the presence of multiple mucosal Cl- exit pathways (Fig. 7) .37 Apical localization of cystic fibrosis transmembrane conductance regulator (CFTR, a chloride channel activated by intracellular cAMP-dependent protein kinases) has been confirmed by electrophysiological, molecular biological, and immunocytochemical methods in the pigmented rabbit conjunctival epithelium.38 39 In our studies, conjunctival Isc was stimulated 60% to 70% above baseline in a sustained manner after the addition of mucosal 8-Br cAMP and remained comparably sensitive to mucosal H2O2 at IC50 as the untreated tissues (Fig 2A) . Furthermore, approximately 30% of the 8-Br cAMP-inducible Isc persisted in conjunctival tissues pretreated with mucosal H2O2 at IC50, suggesting that H2O2-induced oxidant stress may partly inhibit mechanisms of conjunctival cAMP dependent Cl- secretion. In our previous study, when conjunctival epithelial cell layers in primary culture were treated with BSO for 24 hours, a more than 90% depletion of their total cellular GSH level was observed, whereas 80% lower levels of cAMP production were detected after 10 minutes of stimulating cells with forskolin (a cell-permeable diterpenoid that possesses adenylyl cyclase activating properties).40



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  		FIGURE 7. Schematic diagram illustrating how H2O2 may be involved in inhibition of conjunctival active ion transport and the protective role of mucosal GSH. Mucosal application of H2O2 can directly deplete tear fluid GSH generating various ROS. Diffusion of H2O2 into conjunctival epithelial cells can cause a significant depletion of total cellular GSH, generating cytosolic ROS, and leading to direct impairment of ion transporters or their regulatory pathways (i.e., cAMP-dependent Cl- channels) from the cytosolic side. The irreversible results (i.e., loss of conjunctival TEER) of tissue serosal surface oxidation with H2O2 can be attributed to the presence of the Na+/K+-ATPase pump, and the bumetanide-sensitive Na+/K+/2Cl- cotransporter, both of which are essential for conjunctival Isc, and damage to tight junctional complexes. Circles: ion transporters. Parallel dotted lines nested in the cell membrane designate ion channels. Unidirectional arrows: ion, solute, or GSH transport. Bidirectional arrows: the equilibrative diffusion of H2O2. Cylinder designates unknown GSH mucosal efflux transporter. () Tight junctions; ({intcal}) an inhibition of activity or a decrease in level (and paired with ? indicates the possible presence of catalase); () direction of transport; (->) production of ROS.

 
Inhibition of conjunctival Isc of up to 70% with serosal application of 0.5 mM bumetanide, an inhibitor of Na+,K+,2Cl- cotransporter (serosal entry pathway for Cl- into conjunctival cells), has been reported.13 41 The bumetanide-sensitive portion of conjunctival Isc inhibited with mucosal application of H2O2 in this study was apparently greater than the Na+-sensitive fraction of Isc (Fig. 2B) . The extent of Isc decrease afforded with both mucosal H2O2 and serosal bumetanide together was overlapping, whereas the removal of Na+ was additive to further decrease Isc when combined treatments were used (Fig. 2C ; data not shown for Na+-free BRS and serosal bumetanide). The extent of the CFTR contribution to cAMP-regulated Isc in pigmented rabbit conjunctiva needs further elucidation. Chloride appears to enter the pigmented rabbit conjunctiva from the serosal fluid through the Na+,K+,2Cl- cotransport process and to exit into the mucosal fluid through chloride channels, resulting in active Cl- secretion.13 41 Active Cl- secretion in the pigmented rabbit conjunctiva appears to be modulated by multiple mechanisms, one of which is cAMP.37 38 Diminution of cAMP- and bumetanide-dependent Cl- flux across the pigmented rabbit conjunctiva due to oxidative insult may manifest an elevation of Na+ absorption under such conditions. Additional analysis is needed to ascertain fully the relative sensitivity of pharmacological treatments known to modulate conjunctival net Cl- secretion, although these phenomenological studies may give only circumstantial evidence at most.

Investigations of tear fluid on formation of reactive oxygen species (ROS) in the tear specimens from different human hosts showed marked inhibition of hydroxy radical formation, but did not affect superoxide or H2O2 levels.42 The efficacy of GSH when added to the mucosal bathing fluid in preventing (Table 1) or helping recovery (Table 2 , Fig. 3 ) of mucosal H2O2-induced impairment of conjunctival Isc (but not that of serosal H2O2) may be attributable to the highly polarized distribution of chloride12 41 43 and GSH6 secretory machinery in the mucosa of the conjunctival epithelium (Kannan R, et al. IOVS 2002;43:ARVO E-Abstract 905). In addition, the exit of GSH from epithelial cells may be partially coupled and regulated by the parallel secretion of Cl-,5 44 although we do not think CFTR actually conducts GSH. Using an established adenovirus type 5 ocular infection model in pigmented rabbits,45 46 manifesting characteristics of oxidative stress, we have recently described the pharmacological regulation of diminished chloride and fluid secretion,43 as well as significant reduction in GSH secretion, using excised conjunctival tissues from these animals (Kannan R, et al. IOVS 2002;43:ARVO E-Abstract 905).

In our study, the relative ineffectiveness of the stronger reducing agents DTT and 2-mercaptoethanol (compared with the effects of exogenous GSH) in preventing or reversing the damaging effects of H2O2 (Tables 1 and 2) is noted. Partial recovery of Isc in conjunctival tissues pretreated with mucosal H2O2 was achieved by mucosal instillation of mannitol, another known hydroxyl radical scavenger.47 48 An interesting observation was that when tissues were pretreated with ebselen, a small-molecule antioxidant characterized by glutathione peroxidase-mimicking properties,49 the inhibitory effects of mucosal H2O2 on conjunctival Isc were prevented, even though the tissue TEER decreased to a much greater extent than with other antioxidants. At present, we do not know why. By contrast, when GSH-MEE, a zwitterionic analogue of GSH with better diffusion characteristics across cell membranes, was used,50 recovery and protection of conjunctival Isc was achieved with smaller doses compared with those of GSH necessary to achieve similar results. Although limited information exists on the ability of mammalian tears to neutralize H2O2, these findings suggest that the ocular surface and tear film may be endogenously endowed with some antioxidant capacity under normal conditions.

To our knowledge, the specific role of the conjunctiva and that of its mucosal GSH secretion have not been studied for their role in protection from oxidant injury to the ocular surface. Especially the mechanisms underlying the observed irreversible serosal H2O2-mediated damage to factors governing conjunctival Isc may be related to its direct damage of ion-transporting proteins (e.g., Na+,K+-ATPase), or other detrimental effects resulting in a general compromise of the conjunctival epithelial barrier. Chemical modification of the protein residues essential for Na+,K+-ATPase activity, peroxidation of the enzyme-lipid environment, or the effect of lipid peroxidation products on the enzyme’s structure are known to occur with H2O2.51 Furthermore, other reports indicate that the exposure of Madin-Darby canine kidney cells to H2O2 causes a significant derangement of zonula occludens-1 (ZO-1) and b-catenin, resulting in a general reduction of cell adherence and impairment of tight junctions.52

(Extra)cellular Antioxidants
The shorter mucosal t1/2 for H2O2 disappearance may be due to the presence of peroxide-scavenging mechanisms localized in the mucosal surface of rabbit conjunctival tissues. Conjunctival tissues pre-exposed to mucosal 1.5 mM H2O2 for 120 minutes displayed significant intracellular GSH depletion, whereas those with GSH or GSH-MEE pre- or posttreatment preserved at least 100% of their total GSH (Fig. 5) . We noted that 15 µM serosal H2O2 did not cause any changes in total cellular GSH from conjunctival tissues (Fig. 5) . These results suggest that the relationship between mucosal or serosal H2O2-induced changes in Isc and total cellular GSH from conjunctival tissues exposed to these same treatments may be more complex. Similarly, studies in which the response of cultured bovine lens epithelial cells to oxidative challenge was investigated reveal a comparable 44% decrease in intracellular GSH with 1 mM H2O2 treatment.53 As the decrease in total tissue GSH may affect several mechanisms (including the rate of mucosal GSH secretion) and could mediate the changes in conjunctival barrier properties, further studies are needed to investigate the protective role of mucosal GSH and conjunctival GSH secretion under oxidant insult. That exogenous application of GSH esters (mono- and diethyl-ester analogues) increases intracellular GSH content has been established,50 but to our knowledge it has not been directly considered for functional modulation of tissue or cellular ion transport.

Na+,K+-ATPase-Dependent Changes in Isc
Glutathione is known to modulate the activity of several cytoplasmic enzymes and membrane spanning proteins (e.g., transporters).20 54 The action of oxidative agents on membrane channel currents and channel conductance properties has been studied in retinal pigmented epithelial cells, where the application of extracellular GSH abolished the inhibitory effect of H2O2 on membrane channel currents measured in whole-cell recordings. Furthermore, precursors known to increase the intracellular GSH level partially protected these currents from subsequent exposure to harmful levels of H2O2, in a partially glutathione S-transferase-dependent manner.20 In line with these reports, in this study we estimated the amount of functional Na+,K+-ATPase expressed on the serosal side of conjunctival tissues by using a specific inhibitor of Na+,K+-ATPase-dependent transport, ouabain.14 26 In our studies, an 80-minute incubation with 15 nM serosal 3H-ouabain in K+-free BRS did not alter the Isc, whereas in the presence of mucosal or serosal H2O2 at IC50 the specific serosal binding of 3H-ouabain was highly inhibited (Fig. 6) . Mucosal application of ouabain (<=0.5 mM) to pigmented rabbit conjunctival tissues did not influence the Isc, whereas virtually all of the Isc was inhibited at 0.1 mM or more ouabain added serosally.12 Pretreatment of tissues with GSH, GSH-MEE, or ebselen for 60 minutes in the mucosal fluid, or posttreatment with mucosal GSH or GSH-MEE for up to 100 minutes reduced the degree of inhibition in 3H-ouabain binding from 85% to 30%–55% (Fig. 6) . The strong inhibitory effect of serosal H2O2 on the rabbit conjunctival Isc may be related to the characteristics of the active Na+,K+-ATPase-driven short-circuit current in this tissue.12 Conjunctival mucosal GSH appears only to protect from direct mucosal (but not serosal) oxidative insult by H2O2, whereas serosal GSH is not protective in both cases. The actual propagation mechanism of the exclusive mucosal protection with GSH of conjunctival serosal transporters (such as the Na+,K+-ATPase) responsible for the maintenance of normal membrane permeability to ions and solutes is unidentified and requires further investigation.

A hypothetical scheme describing possible mechanisms of an H2O2-induced decrease in conjunctival ion transport is presented in Figure 7 , which is based on previous reports on GSH transport6 and the localization of relevant conjunctival ion transporters.13 37 38 39 Mucosal application of H2O2 can directly deplete the steady state tear fluid GSH level, generating various ROS on the mucosal side of conjunctival tissues. In addition, H2O2 can rapidly equilibrate across tissue membranes, diffusing into conjunctival epithelial cells where we observed a significant depletion of total GSH (Fig. 5) . A decrease in cellular GSH is accompanied by an increase in ROS, which may lead to further epithelial cell oxidative damage. An increase in cytoplasmic ROS or H2O2 may interfere with the function of various ion transporters by blocking regulatory pathways (i.e., 8-Br cAMP results from Fig. 2A ) or triggering the sequestering and/or destruction of oxidized ion transporters (i.e., decrease in specific ouabain binding to Na+,K+-ATPase in Fig. 6 ). The irreversible decreases in Isc by serosal application of H2O2 may be attributable to the oxidant-caused derangement in the function or number of the Na+,K+-ATPase pump and the bumetanide-sensitive Na+,K+,2Cl- cotransporter.13 In addition, the role of catalase in conjunctival tissues is mostly unclear.

In summary, our results show that the presence of mucosal GSH is crucial for the maintenance of proper ion transport activity in isolated pigmented rabbit conjunctival tissues. Protection by mucosally applied GSH (or analogues) of functional Na+,K+-ATPases in the serosal membranes may be useful in maintaining the physiological activity of conjunctiva under oxidative stress. Conjunctival net GSH secretion may be the primary method of counteracting mucosally generated peroxides present within the immediate microenvironment of the tear film in contact with this tissue. Glutathione may be important in the modulation of cellular responses that lead to the processing of oxidized, damaged, and nonfunctional membrane proteins found in age-related ocular diseases under oxidant stress.


   Acknowledgements
 
The authors thank Stephanie Patnode (Department of Biomedical Engineering, University of Southern California, Los Angeles, CA) for exceptional technical assistance and for lending excellent biomedical engineering expertise in programming the software for the digital data acquisition and subsequent analysis of electrophysiological measurements; Austin K. Mircheff (Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, Los Angeles, CA) for helpful discussions.


   Footnotes
 
Supported in part by National Eye Institute Grants EY11135 (RK) and EY12356 (VHLL), National Heart, Lung, and Blood Institute Grants HL38658 (K-JK) and HL64365 (K-JK, VHLL), and National Institute of General Medical Sciences Grant GM28673 (RAF). HJG was awarded an American Foundation for Pharmaceutical Education predoctoral fellowship and Town & Gown postgraduate scholarships from the University of Southern California.

Submitted for publication May 2, 2003; revised July 1, 2003; accepted July 9, 2003.

Disclosure: H.J. Gukasyan, None; K.-J. Kim, None; R. Kannan, None; R.A. Farley, None; V.H.L. Lee, None

Presented at the annual meetings of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 2002 and 2003.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Vincent H. L. Lee, University of Southern California, School of Pharmacy, Department of Pharmaceutical Sciences, 1985 Zonal Avenue, PSC 704, Los Angeles, CA 90089-9121; vincentl{at}hsc.usc.edu.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

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