Specialized Protective Role of Mucosal Glutathione in Pigmented Rabbit Conjunctiva

Abstract

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.

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 (I_sc), 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 I_sc 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-α1-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 (K _i ∼16 nM) and a low-affinity (K _i ∼4.2 μM) site. ¹⁴ Measurements of tissue I_sc 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 I_sc in rat tracheal ¹⁵ or alveolar epithelial cell monolayers cultured on permeable support, ¹⁶ as well as in several ocular tissues, including those of the amphibian corneal epithelium ¹⁷ 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 I_sc 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

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 I_sc (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-I_sc endpoints (e.g., H₂O₂ levels in bathing fluids, total tissue GSH level, and ³H-ouabain binding) described in the following sections were performed when I_sc reached the steady state level after each treatment. 

Conjunctival Studies of I_sc on H₂O₂ Dose-Response and Treatment with Other Reagents

The effect of hydrogen peroxide on conjunctival I_sc 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 I_sc were expressed as a percentage of initial I_sc, 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  

\[Y{=}\mathrm{bottom}{+}(\mathrm{top}{-}\mathrm{Bottom})/(1{+}10^{\mathrm{{[}(log\ IC}_{50}{-}X){\times}\mathrm{hill\ slope{]}}})\]

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

To determine ion transport mechanisms that may be influenced during inhibition of conjunctival I_sc with mucosal (or serosal) H₂O₂ at IC₅₀, conjunctival tissues were treated sequentially with H₂O₂ followed by a maneuver that is known to modulate I_sc 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 I_sc, or the remaining I_sc after the specific maneuver, which partially alters a component thereof. To determine whether the H₂O₂-induced decreases in conjunctival I_sc 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 I_sc 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 I_sc. 

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 (l_max, 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 (t_1/2) 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  

\[\mathrm{log{[}}S{]}{=}(-k/2.3)\ {\cdot}\ \mathrm{t}\ {+}\ \mathrm{log{[}}S_{0}{]}\]

where [S] represents the concentration of H₂O₂ at various time points t, k denotes the reaction (i.e., decomposition) rate, and S ₀ 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 t_1/2, and the reaction rate k can be estimated from the equation 0.693/k = t_1/2. 

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 I_sc 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 I_sc (as described earlier). After the H₂O₂ effect reached steady state I_sc, 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 × 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 I_sc 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 I_sc is inhibited was reported by our laboratories to be approximately 20 μM with a t_1/2 for I_sc 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 I_sc 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 I_sc 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

Effect of Mucosal or Serosal H₂O₂ on Conjunctival I_sc

Figure 1 shows the decreases in I_sc as a percentage of its value at t = 0 in response to increasing doses of H₂O₂. At physiological concentrations of H₂O₂ found within cells ^{27 28} or in other avascular intraocular compartments ² (0–100 μM), I_sc did not change significantly by mucosal application of H₂O₂. Hydrogen peroxide afforded approximately two orders of magnitude difference in 50% inhibition of conjunctival I_sc from the mucosal versus serosal side. At concentrations of H₂O₂ that elicited significant decreases in I_sc, steady state levels were reached within 15 minutes of H₂O₂ application. 

Effect of 8-Br cAMP, Bumetanide, and Na⁺-free BRS on Mucosal H₂O₂-Induced I_sc 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 I_sc, 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 I_sc increased above baseline by 66%. Mucosally applied H₂O₂ at maximum 8-Br cAMP effect drastically inhibited the I_sc to 15% below the initial baseline (Fig. 2A) . For the reverse treatment sequence, I_sc 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. 

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

Effect of GSH Treatment on H₂O₂-Induced I_sc Impairment

The I_sc 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 I_sc from 1.5 mM mucosal H₂O₂-induced conjunctival I_sc decrease. When used at 1 and 2.5 mM, GSH was ineffective during mucosal pre- or posttreatments in recovery of I_sc within the time course tested, whereas at 5 mM it was ineffective in I_sc 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, I_sc or TEER did not change significantly from baseline. In contrast, 0.1 mM ebselen pretreatment led to decreases in I_sc 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 I_sc. The final I_sc 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 I_sc 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 I_sc. 

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 I_sc after 15 minutes of treatment (Fig. 3) . The decrease in I_sc 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 I_sc (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 I_sc (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 I_sc 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 I_sc 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 I_sc or TEER (Table 2) . 

All mucosal or serosal treatments were essentially ineffective in reversing the insult of serosal H₂O₂ at IC₅₀ on conjunctival I_sc or TEER. Furthermore, restoration of I_sc 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 I_sc 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 × 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 × 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 × 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. 

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 I_sc 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) . 

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 I_sc 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. 

Discussion

In this study, the effect of H₂O₂ on I_sc 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 I_sc

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 I_sc 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 I_sc 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 I_sc 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 H₂O₂ ²⁹ 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 humor ³² and lenses ³³ 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 mM ³⁴ ), through a reaction with riboflavin (detected in ocular surface tissues of the rabbit ³⁵ ), 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 t_1/2 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 I_sc

Conjunctival epithelial cells display an active mucosal secretion of Cl⁻, a phenomenon estimated to account for approximately 70% of I_sc 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 I_sc 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 I_sc 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  

Inhibition of conjunctival I_sc 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 I_sc inhibited with mucosal application of H₂O₂ in this study was apparently greater than the Na⁺-sensitive fraction of I_sc (Fig. 2B) . The extent of I_sc decrease afforded with both mucosal H₂O₂ and serosal bumetanide together was overlapping, whereas the removal of Na⁺ was additive to further decrease I_sc 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 I_sc 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 I_sc (but not that of serosal H₂O₂) may be attributable to the highly polarized distribution of chloride ^{12 41 43} and GSH ⁶ 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 I_sc 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 I_sc 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 I_sc 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 I_sc 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 t_1/2 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 I_sc 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 I_sc

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 I_sc, 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 I_sc, whereas virtually all of the I_sc 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 I_sc 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 transport ⁶ 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 I_sc 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. 

 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.

 

Figure 1.

 

Dose-response of conjunctival I_sc to various mucosal and serosal concentrations of H₂O₂ in normal BRS. The data show the percentage decreases in I_sc 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, I_sc was unaffected. The serosal aspect of excised conjunctiva was more sensitive by two orders of magnitude to the H₂O₂-induced decrease in I_sc. The control I_sc was approximately 10 μA/cm². Data are the mean ± SEM of results in three to four tissues.

Figure 2.

 

Time course of I_sc 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 I_sc 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 I_sc 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 I_sc 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 I_sc 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.

Table 1.

 

Effect of Mucosal Pretreatment on Changes in Conjunctival I_SC by Mucosally Applied H₂O₂

±Pretreatment	±H2O2	ISC (μA/cm2)	TEER (kΩ×cm2)
						Initial	Final
−	−	9.80 ± 0.20	1.46 ± 0.07
−	+	2.61 ± 0.49, †		0.30 ± 0.01, ∥
+GSH	+	9.15 ± 0.38, ‡		0.95 ± 0.05
+GSH-MEE	+	8.85 ± 0.29, ‡		0.97 ± 0.04
+Ebselen	+	5.20 ± 0.52, ‡ , §		0.20 ± 0.01, ∥

Figure 3.

 

Time course of I_sc 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.

Table 2.

 

Effect of Mucosal Posttreatments on Changes in Conjunctival I_SC by Mucosally Applied H₂O₂

±H2O2	±Posttreatment	ISC (μA/cm2)	TEER (kΩ×cm2)
						Initial	Final
−	−	8.14 ± 0.10	1.54 ± 0.07
+	−	2.85 ± 0.36*		0.30 ± 0.01, ‡
+	+BRS wash	1.35 ± 0.10		0.29 ± 0.01, ‡
+	+GSH	6.10 ± 0.34, †		0.90 ± 0.01
+	+GSH-MEE	7.62 ± 0.36, †		1.00 ± 0.10
+	+DTT	1.79 ± 0.44		0.23 ± 0.01, ‡
+	+2-mercaptoethanol	1.95 ± 0.53		0.15 ± 0.01, ‡
+	+D-mannitol	3.95 ± 0.24, †		0.91 ± 0.05
+	+sucrose	2.99 ± 0.34		0.32 ± 0.03, ‡
+	+ebselen	2.20 ± 0.12		0.20 ± 0.09, ‡

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 × 10⁻³ (r ² = 0.97, ▴) and −3.4 × 10⁻³ (r ² = 0.96, □) for H₂O₂ consumption from mucosal and serosal fluids of normal BRS, respectively, were estimated. A t_1/2 for H₂O₂ of approximately 25 and 143 minutes in respective bathing fluids of the conjunctiva was estimated using the relation, t_1/2 = 0.693/(−2.3 × slope). Data are the mean ± SEM of results in three to four tissues.

Figure 5.

 

Changes in total cellular GSH level in excised conjunctiva were assayed after the maximum effect on I_sc. 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%. †P < 0.05 vs. BRS control. ‡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.

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 I_sc) 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 I_sc 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 (□) and nonspecific (▪) ³H-ouabain binding level with the total across all categories. †P < 0.05 versus control. ‡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.

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 I_sc, 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; (⊺) an inhibition of activity or a decrease in level (and paired with ? indicates the possible presence of catalase); (➟) direction of transport; (→) production of ROS.

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.