Hydrogen peroxide inhibits cAMP-induced Clminus secretion across colonic epithelial cells

Michael D. DuVall1, Yi Guo1, and Sadis Matalon1,2,3

Departments of 1 Anesthesiology, 2 Physiology and Biophysics, and 3 Pediatrics, University of Alabama, Birmingham, Alabama 34294

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We examined the effects of H2O2 on Cl- secretion across human colonic T84 cells grown on permeable supports and mounted in modified Ussing chambers. Forskolin-induced short-circuit current, a measure of Cl- secretion, was inhibited in a concentration-dependent fashion when monolayers were pretreated with H2O2 for 30 min (30-100% inhibition between 500 µM and 5 mM). Moreover, H2O2 inhibited 76% of the Cl- current across monolayers when the basolateral membranes were permeabilized with nystatin (200 µg/ml). When the apical membrane was permeabilized with amphotericin B, H2O2 inhibited the Na+ current (a measure of Na+-K+-ATPase activity) by 68% but increased the K+ current more than threefold. In addition to its effects on ion transport pathways, H2O2 also decreased intracellular ATP levels by 43%. We conclude that the principal effect of H2O2 on colonic Cl- secretion is inhibitory. This may be due to a decrease in ATP levels following H2O2 treatment, which subsequently results in an inhibition of the apical membrane Cl- conductance and basolateral membrane Na+-K+-ATPase activity. Alternatively, H2O2 may alter Cl- secretion by direct action on the transporters or alterations in signal transduction pathways.

cystic fibrosis transmembrane conductance regulator; short-circuit current; T84 cells; sodium-potassium-adenosinetriphosphatase; potassium channels

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

REACTIVE OXYGEN AND nitrogen species are involved in a variety of intestinal disorders and dysfunction, including ischemia-reperfusion and inflammatory bowel disease. These species are generated by inflammatory cells and may also be produced intracellularly by gastrointestinal cells from incomplete reduction of oxygen at the mitochondria, or by various cytoplasmic sources (15). Kurose and Granger (22) showed that hydrogen peroxide (H2O2) and hydroxyl radicals (· OH), generated by xanthine oxidase during reperfusion of the ischemic gut, damage the intestinal mucosa. In addition, inflammatory cells (monocytes, eosinophils, and interstitial macrophages) from patients with Crohn's or inflammatory bowel disease generate significantly higher levels of reactive species than inflammatory cells isolated from normal humans (36, 44, 45).

H2O2, formed by the dismutation of superoxide anions or the two-electron reduction of oxygen, plays an important role in the initiation and propagation of epithelial inflammatory injury. H2O2 per se can react with lipids and sulfhydryls to inactivate key enzymes, albeit at a slow rate (30). However, because of its relatively long half-life and lipophilic nature, H2O2 can also enter cells, where it can react with transition metals, such as iron, to yield the extremely reactive · OH. In addition, myeloperoxidase, which is released by neutrophils, catalyzes the formation of hypochlorous acid (HOCl), a powerful oxidizing and chlorinating agent (16). Moreover, recent studies indicate that HOCl may interact with nitrite, the stable by-product of nitric oxide metabolism, to form potent chlorinating and nitrating species that may damage key cellular components (13). For these reasons, there is considerable interest in identifying the effects and mechanisms by which H2O2 alters ion transport across the intestinal epithelium.

Cl- secretion across the intestinal epithelium plays an important role in fluid homeostasis and mucosal hygiene. The human adenocarcinoma cell line T84 has been widely used as a model system for the study of Cl- secretion across epithelial monolayers. This process requires the coordinated action of several transporters (see Ref. 2 for review). First, the Na+-K+-ATPase localized to the basolateral membrane is responsible for establishing and maintaining Na+ and K+ gradients across the cell membrane. Second, basolateral K+ channels act to recycle K+, brought into the cell through the activity of the Na+-K+-ATPase, back into the serosal space. Moreover, these channels are important in providing a sustained electrical driving force necessary to maintain Cl- secretion. Third, the Na+-K+-2Cl- cotransporter loads Cl- into the cells above its electrochemical equilibrium by coupling Cl- transport to Na+. Last, the apical membrane Cl- channel conducts Cl- out of the cell down its electrochemical gradient.

Previous studies using T84 cells, grown to confluence and mounted in Ussing chambers, have shown that addition of H2O2 to the apical or basolateral sides increased Cl- secretion in a transient manner. More importantly, H2O2 attenuated the large Cl- secretory current produced by agents that increase intracellular cAMP (27). However, the specific transport pathways responsible for the inhibitory effect of H2O2 on Cl- secretion were not identified. Herein we report on a series of experiments designed to clarify which epithelial transport processes were affected by H2O2, giving rise to the observed inhibition of Cl- secretion across intact T84 monolayers. Using the pore-forming antibiotics nystatin and amphotericin B to permeabilize the basolateral and apical membranes, respectively, we were able to isolate currents and assess the effects of H2O2 on 1) the apical membrane Cl- conductance, 2) the basolateral membrane Na+-K+-ATPase activity, and 3) the basolateral membrane K+ conductance. Our results indicate that H2O2 inhibits Cl- secretion by decreasing the apical plasma membrane Cl- conductance and the activity of the Na+-K+-ATPase. Furthermore, the results suggest that these effects may be due to a fall in intracellular ATP concentration.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Culture

T84 cells (obtained from the American Type Culture Collection, Manassas, VA) were cultured in a mixture of DMEM and Ham's F-12 (50:50) supplemented with 10% fetal bovine serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin and grown in plastic tissue culture flasks at 37°C and 5% CO2. When monolayers were ~90% confluent, the cells were subcultured onto Millicell HA culture inserts (Millipore, Bedford, MA) with an area of 0.6 cm2. Experiments were conducted on confluent monolayers 8-12 days after culture onto the permeable supports. Cell passages between 31 and 49 were used for these studies.

Transepithelial Transport Studies

All transport experiments were conducted under short-circuit conditions. Confluent monolayers were mounted into modified Ussing chambers and connected to a transepithelial voltage clamp (Warner Instruments, Hamden, CT) that allowed continuous measurement of the short-circuit current (Isc), an indicator of transepithelial Cl- secretion. Transepithelial resistance (Rt) was calculated by measuring the Isc resulting from 5-s square voltage pulses (2 or 4 mV) imposed across the monolayer every 1 min. The standard composition of the apical and basolateral bathing solutions was (in mM) 145 Na+, 5 K+, 125 Cl-, 1.2 Ca2+, 1.2 Mg2+, 25 HCO-3, 3.3 H2PO-4, 0.8 HPO2-4, and 10 glucose (pH 7.4). Experimental design required modification of the bathing solution compositions for specific experiments, and these changes are indicated. All solutions were gassed with 95% O2-5% CO2 at 37°C.

After mounting of the filters containing T84 cells into the Ussing chambers, the Isc was allowed to stabilize before each experiment (10-15 min). In our initial experiments, we increased intracellular cAMP levels by the addition of forskolin (10 µM) to both sides of the monolayers, which caused the Isc to increase. We added H2O2 (500 µM) to both bathing solutions at the peak of the forskolin response and examined its effect on the stimulated Isc. To evaluate the effects of H2O2 on basal Cl- secretion and on subsequent cAMP-stimulated secretion, we treated both sides of the mounted T84 monolayers with H2O2 in the absence of forskolin. Responses to forskolin treatment 30 min later were then measured as a change in Isc, determined as the difference in area under the Isc trace 10-15 min before and after treatment periods divided by the time interval. Measurements were repeated in the presence of catalase, which catalyzes the conversion of H2O2 to water. In a separate set of experiments, the effects of H2O2 on the forskolin-induced Isc were measured and compared with those from monolayers bathed in 2-deoxyglucose (5 mM), a compound that significantly decreased intracellular ATP concentration (see Effect of H2O2 on Intracellular ATP Levels).

We evaluated the role of intracellular Ca2+ in the H2O2 response by preincubating monolayers with 50 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM for 30 min. Monolayers were then treated with either H2O2 (500 µM) or an equivalent volume of water for an additional 30 min. Last, monolayers were treated with forskolin (10 µM). The current responses were measured as before.

To evaluate the specific effects of H2O2 on electrogenic ion transport processes involved in Cl- secretion across mounted monolayers, we used the pore-forming antibiotics amphotericin B and nystatin to selectively permeabilize the apical and basolateral membranes, respectively. These methods are described below.

Apical membrane Cl- conductance. To evaluate whether H2O2 had any direct effects on the apical membrane Cl- conductance, monolayers were mounted in Ussing chambers under short-circuit conditions in the presence of an apical-to-basolateral (126:6 mM) Cl- gradient (sodium gluconate substituted for NaCl in the serosal medium). Because the cystic fibrosis transmembrane conductance regulator (CFTR) protein will conduct Cl- in either direction, the Cl- concentration gradient was set to drive Cl- movement from the apical to the basolateral side of the monolayers to eliminate the contributions of the Na+-K+-2Cl- cotransport and Na+-K+-ATPase to the Isc. After the Isc reached steady state, the basolateral membrane was permeabilized by the addition of nystatin (200 µg/ml). Forskolin was then added to the apical and basolateral sides of the monolayers to activate CFTR. Under these conditions, the Isc represents the Cl- current (ICl) as Cl- moves down its concentration gradient through the CFTR Cl- channels in the apical plasma membrane.

Na+-K+-ATPase activity. The effect of H2O2 on Na+-K+-ATPase activity was examined in monolayers mounted in Ussing chambers bathed with medium in which NaCl was replaced by N-methyl-D-glutamine chloride (NMDG-Cl), such that the final bath Na+ concentration was 25 mM on both sides of the monolayers. The apical membrane was then permeabilized by addition of amphotericin B (10 µM) to the apical bathing solution. Under short-circuit conditions, the resulting current is due to the transport of Na+ across the basolateral membrane by the Na+-K+-ATPase (INa).

The concentration response relationship of the pump current for bath Na+ was evaluated by initially bathing both sides of the monolayers mounted in Ussing chambers with Na+-free Ringer solution (NaCl replaced with NMDG-Cl and NaHCO3 replaced with choline bicarbonate). Amphotericin B was then administered to the apical bathing solution, and the INa was continuously recorded. Thereafter, the Na+ concentration was incrementally increased by removing bathing medium from both sides of the monolayers and replacing it with equal volumes of normal Ringer. Thus bath Na+ concentration was incrementally increased over a range from 0 to 100 mM without affecting the concentrations of other ions. Current responses were fitted with the Hill equation. The maximal current (Vmax) and the Hill coefficient were subsequently compared between control and H2O2-treated monolayers.

In separate experiments, INa was also recorded in monolayers bathed with 5 mM 2-deoxyglucose and 25 mM Na+ (NaCl replaced with NMDG-Cl). The results were compared with those from control and H2O2 (500 µM)-treated monolayers.

Basolateral membrane K+ conductance. To evaluate the effect of H2O2 on the basolateral K+ conductance of T84 cells, we mounted monolayers in Ussing chambers in the presence of an apical-to-basolateral K+ gradient (80:5 mM), while the Na+ concentration was maintained at 25 mM on both sides of the monolayers. NaCl in the apical bathing solution was replaced with KCl, and NaCl in the serosal bathing solution was replaced with NMDG-Cl. After ~15 min, H2O2 or water was added bilaterally to the monolayers and the Isc was measured for 30 min. Ouabain (100 µM) was then added to the serosal bath to inhibit the Na+-K+-ATPase, and amphotericin B (10 µM) was added to the apical bath to permeabilize the apical plasma membrane. The resulting Isc is due to the movement of K+ through channels in the basolateral membrane (IK).

We also evaluated the immediate effects of H2O2 on the basolateral K+ conductance by first treating monolayers with ouabain and amphotericin B (added to the basolateral and apical baths, respectively) in the presence of a K+ gradient. At the peak of the amphotericin-induced response, H2O2 was applied bilaterally to the bath while continuously measuring the IK.

H2O2 Measurement

We measured H2O2 concentrations in each compartment of the Ussing chambers as previously described (3). Briefly, 200-µl aliquots of bathing medium were removed and added to 800 µl of potassium phosphate solution (pH 7.0) containing 3,000 units of horseradish peroxidase, 1.5 mM 4-aminoantipyrine, 0.11 M phenol, and 100 µM allopurinol. The H2O2 concentration was then calculated by measuring the sample absorbance at 510 nm (extinction coefficient epsilon M = 6.58 mM-1 · cm-1).

Lactate Dehydrogenase Release Assay

To assess the cytotoxic potential of H2O2 on T84 cells, we measured lactate dehydrogenase (LDH) release into the medium. Confluent monolayers were treated with 5 mM H2O2 added to both mucosal and serosal bathing solutions (45 min at 37°C). Results were compared with LDH measurements made from monolayers in the absence of H2O2. At the end of 45 min, the medium inside the chambers was removed and the cells were lysed with 6.5 ml of 1% (wt/vol) Triton X-100 in our Ringer solution. LDH activity in the medium and cell lysate was measured by the method of Bergmeyer and Bernt (5). Results were reported as the percentage of LDH released into the medium [(LDH in the medium)/(LDH in the medium + LDH in the lysate)].

ATP Measurement

T84 monolayers were incubated in H2O2 (500 µM) or 2-deoxyglucose (5 mM) for 30 min. After that, ATP levels were measured by a bioluminescence assay that uses luciferin as a substrate in the presence of firefly luciferase (Sigma, St. Louis, MO). Emitted light was measured in a Monolight 20-10 luminometer. When ATP is the limiting reagent, the light emitted is proportional to the ATP present.

Chemicals

Forskolin, BAPTA-AM, nystatin (Mycostatin), and amphotericin B were purchased from Calbiochem (La Jolla, CA). All other drugs and chemicals were purchased from Sigma. Drugs were introduced into the bathing solution in small volumes (>1% of final volume) from concentrated stock solutions.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

H2O2 Inhibits Cl- Secretion Across T84 Monolayers

Measurements across T84 monolayers under basal conditions revealed a small Isc (~3 µA/cm2) suggestive of a low level of Cl- secretion. When forskolin (10 µM) was added to both sides of the monolayers, the Isc rapidly increased from 4 to 46 µA/cm2 and remained significantly elevated for over 50 min. H2O2 (500 µM) added to both bathing solutions at the peak of the forskolin response increased the Isc further (20 ± 12 µA/cm2; n = 6), but only transiently. This was followed by a gradual decrease in secretion that dropped to <50% of that observed in monolayers that received forskolin alone. Moreover, catalase (1,000 U/ml), which catalyzes the conversion of H2O2 to water, added to both sides of the monolayers blocked the effects of H2O2 (Fig. 1).


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Fig. 1.   Short-circuit current (Isc) across monolayers of T84 cells mounted in an Ussing chamber. Addition of forskolin (Forsk; 10 µM) to both apical and basolateral baths resulted in a large sustained increase of Isc (control). Unlabeled arrow indicates point at which water, H2O2, or catalase followed by H2O2 was added to bath. Addition of H2O2 (500 µM) to both sides of monolayer resulted in a large initial increase of Isc, followed by a decrease within 10 min. This effect of H2O2 on Isc was totally blocked in presence of catalase (1,000 U/ml). This typical result was repeated at least 3 times.

When H2O2 (500 µM) was added to monolayers in the absence of forskolin, the Isc increased by 3 ± 1 µA/cm2 (n = 12), but only transiently. Subsequent treatment with forskolin 30 min later increased the Isc from 6 to 33 µA/cm2. This response was ~30% less than that observed in control monolayers (Fig. 2 and Table 1). Moreover, data shown in Table 1 also indicate that Rt values and LDH release into the medium were not different between water controls and H2O2-treated monolayers. This suggested that the effects of H2O2, at concentrations used in this study, were not due to cytotoxicity.


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Fig. 2.   Effect of H2O2 on forskolin-induced Cl- secretion. Addition of H2O2 (500 µM) resulted in a transient small increase in Isc lasting ~5 min. Monolayers treated with H2O2 had an attenuated response to forskolin (10 µM; added to both sides of monolayers) 30 min later. Control monolayers were treated with an equivalent volume of water. This typical result was repeated at least 3 times.

                              
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Table 1.   Effect of H2O2 on forskolin-induced Cl- secretion

The inhibitory effect of H2O2 on forskolin-induced Cl- secretion was concentration dependent with an IC50 of ~700 µM H2O2 (estimated from a linear regression between 100 and 1,000 µM, r = 0.97; Fig. 3). At 500 µM, H2O2 inhibited ~40% of the forskolin-induced secretion, whereas inhibition was complete at 5 mM. After a bolus addition of 500 µM H2O2, the concentration of H2O2 in the bathing solution dropped to 200 µM over 30 min. When monolayers were pretreated with aminotriazole (20 mM), an irreversible inhibitor of catalase, the inhibitory effect of H2O2 was potentiated. Under these conditions, a 500 µM bolus produced a >90% block of forskolin-induced secretion (Fig. 3). This was not significantly different from the inhibition produced by 5 mM H2O2 alone.


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Fig. 3.   Concentration-dependent inhibitory effects of H2O2 on steady-state forskolin-induced increase in Isc (Delta Isc) across T84 monolayers. H2O2 IC50, calculated from a linear regression of values between 100 and 1,000 µM, was 700 µM (r2 = 0.97). Pretreatment of T84 monolayers with aminotriazole (ATZ; 20 mM), an inhibitor of endogenous catalase, greatly enhanced H2O2 effect. Values are means ± SE. Numbers in parentheses are n at each concentration. * P < 0.05 vs. control (t-test).

Effects of H2O2 on Apical Membrane Cl- Conductance and Basolateral Na+-K+-ATPase Activity and K+ Conductance

Apical membrane Cl- conductance. Nystatin, added to the basolateral bathing solution of control monolayers (n = 5), increased the ICl by 2 ± 1 µA/cm2. This likely reflects a constitutively active apical plasma membrane Cl- conductance. Subsequent bilateral addition of forskolin further increased the ICl by 102 ± 17 µA/cm2 (the current is directed downward, reflecting the apical-to-basolateral direction of the Cl- gradient). When monolayers were first treated with H2O2 (500 µM; n = 5) for 30 min, nystatin increased the ICl by 6 ± 2 µA/cm2, which was not different from controls. However, the forskolin-induced increase in ICl was only 24% that of controls (24 ± 5 µA/cm2). These results, shown in Fig. 4, indicate that H2O2 inhibited the apical membrane Cl- conductance through CFTR channels.


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Fig. 4.   H2O2 inhibited Cl- current (ICl) across permeabilized T84 monolayers. A: ICl across T84 cell monolayers mounted in Ussing chambers measured in the presence of an apical-to-basolateral Cl- concentration gradient (125:6 mM) following addition of nystatin (Nyst; 200 µg/ml) to basolateral bathing solution. Downward deflection of ICl in these traces is due to flow of Cl- from apical to basolateral solution. ICl was significantly decreased in monolayers treated for 30 min with H2O2 (500 µM). Control monolayers were treated with an equivalent volume of water. B: forskolin-induced ICl (Delta ICl) following nystatin permeabilization was significantly decreased in presence of H2O2. Values are means ± SE; n = 3 and 5 for control and H2O2, respectively. * P < 0.05 vs. control (unpaired t-test).

Na+-K+-ATPase activity. In bathing solutions containing 25 mM Na+, the addition of amphotericin (10 µM) to the apical bathing solution increased the INa from 1 ± 1 to 18 ± 3 µA/cm2. Under these conditions, the amphotericin-induced INa was completely inhibited by 100 µM ouabain (data not shown). When monolayers were first treated with H2O2 (500 µM) for 30 min, the amphotericin-induced INa was only ~41% of that seen in control monolayers (1 ± 1 to 7 ± 2 µA/cm2). A representative experiment is shown in Fig. 5A. The relationship between Na+-K+-ATPase and the Na+ concentration was fitted to the Hill equation with a Hill coefficient of 0.9, a Michaelis-Menten constant (Km) of 21 mM, and a Vmax of 47 ± 4 µA/cm2 in control monolayers (n = 4). However, after H2O2 treatment, the Hill coefficient increased to 2.0, Km was 17 mM, and Vmax was reduced to 15 ± 1 µA/cm2 (n = 4). These data suggest that H2O2 increased the cooperativity of the Na+-K+-ATPase for Na+ binding but decreased the efficiency of the Na+-K+-ATPase in Na+ transport. These results are shown in Fig. 5B.


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Fig. 5.   H2O2 inhibited Na+-K+-ATPase activity in T84 cell monolayers. A: Na+ current (INa) across T84 cell monolayers mounted in Ussing chambers measured in presence of 25 mM Na+ (NaCl replaced with KCl) on both sides of monolayer following addition of amphotericin B (Ampho; 10 µM) to apical bathing solution. Pretreatment with H2O2 (500 µM) for 30 min significantly reduced INa response following amphotericin B. Control monolayers were treated with an equivalent volume of water. This typical result was repeated at least 3 times. B: monolayers treated with H2O2 (500 µM) had significantly lower maximal current values than were found in control monolayers. Inset: Hill plot of INa values depicting shift in Hill coefficient from 0.9 to 2.0 under control (open symbols) and H2O2-treated (filled symbols) conditions, respectively. Values are means; n = 4.

Basolateral membrane K+ conductance. When amphotericin B was added to the apical bath in the presence of an apical-to-serosal K+ gradient (80:5 mM) and basolateral ouabain (50 µM), the IK immediately began to increase and reached maximal levels within 15 min (2.3 ± 0.4 to 26.8 ± 7.3 µA/cm2; n = 6). As shown in Fig. 6, when monolayers were first treated with H2O2 (500 µM) for 30 min, the amphotericin B-induced IK was more than three times greater than that of control monolayers (4.6 ± 1.0 to 86.1 ± 20.2 µA/cm2; n = 6). Both the amphotericin- and H2O2-stimulated IK were inhibited ~80% by the application of Ba2+ to the serosal bath. When H2O2 was applied bilaterally to the bath at the peak of the amphotericin-induced response, IK increased by an additional 50% (12.2 ± 0.9 to 18.1 ± 2.2 µA/cm2; n = 8) within 3-5 min (Fig. 7).


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Fig. 6.   H2O2 increased K+ current (IK) across permeabilized T84 monolayers. A: IK across T84 cell monolayers measured in presence and absence of H2O2 (500 µM; for 30 min). Amphotericin B (10 µM) was added to apical bath after ouabain (100 µM) was added to basolateral bath. This typical result was repeated at least 3 times. B: IK following amphotericin B treatment (Delta IK) was significantly increased by H2O2 (500 µM). Values are means ± SE; n = 6. * P < 0.05 vs. control (unpaired t-test).


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Fig. 7.   H2O2 acutely increased IK across permeabilized T84 monolayers. H2O2 was added to both bathing solutions at peak of amphotericin-induced IK. A typical experiment is shown. Inset: summary of IK response before and after H2O2 (500 µM) treatment. Values are means ± SE; n = 8. * P < 0.05 vs. basal; ** P < 0.05 vs. basal and amphotericin treated (paired t-test).

Effect of H2O2 on Intracellular ATP Levels

Relative intracellular ATP levels were measured in control (n = 6) and H2O2 (500 µM; n = 4)-treated T84 monolayers. The ATP levels in cells treated with H2O2 were only 57% of the levels found in control (3.0 ± 0.2 and 1.7 ± 0.1 relative light units in control and H2O2-treated monolayers, respectively).

As shown in Fig. 8, 2-deoxyglucose (5 mM) significantly decreased intracellular ATP concentration, Isc, and INa to 42% (n = 6), 21% (n = 14), and 56% (n = 8) of the values measured in control monolayers, respectively. However, none of these levels was significantly different from that measured in H2O2-treated monolayers.


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Fig. 8.   Summary of effects of 2-deoxyglucose (2-dg) on ATP concentration ([ATP]), Isc, and INa. Monolayers were incubated with either 2-deoxyglucose (5 mM) or H2O2 (500 µM) for 30 min. [ATP], Isc, and INa were then measured and compared with values from untreated monolayers. Values are means ± SE. Numbers in parentheses are n for each condition. * P < 0.05 vs. control (Tukey test).

Role of Intracellular Ca2+ on H2O2-Induced Inhibition of cAMP-Stimulated Secretion

As shown in Fig. 9, when monolayers were incubated with BAPTA-AM (50 µM; bilateral bathing solutions), the Isc responses were identical for control and H2O2-treated monolayers. Under these conditions, the H2O2-induced Isc transient was markedly reduced (0.1 ± 0.0 µA/cm2; n = 8) and the forskolin-induced Isc responses were 41.8 ± 10.3 µA/cm2 (n = 4) and 45.2 ± 7.1 µA/cm2 (n = 8) for control and H2O2-treated monolayers, respectively.


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Fig. 9.   Inhibitory effects of H2O2 were blocked by BAPTA-AM pretreatment. Monolayers were incubated with BAPTA-AM (50 µM) for 30 min and then incubated for an additional 30 min with H2O2 (500 µM) or water before administration of forskolin (10 µM). Reported Isc values were made 30 min after BAPTA-AM (basal) and H2O2 (500 µM; Water/H2O2) and 15 min after forskolin (10 µM). Values are means ± SE; n = 4 for -H2O2 and n = 8 for +H2O2.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

H2O2 has been widely studied as a potential regulator of intestinal epithelial function. The large number of activated phagocytes commonly found with ulcerative and granulomatous colitis (34) may secrete millimolar concentrations of H2O2 in close proximity to the colonocytes (38). The focus of several recent studies has been to identify the role H2O2 plays in modulating epithelial ion transport function, specifically Cl- secretion. H2O2 was shown to stimulate Cl- secretion across the intact rat colonic epithelium (19, 37). However, this response was determined to be due to the release of prostaglandins that subsequently acted on submucosal neurons to stimulate the release of neurotransmitters, rather than directly on the epithelium.

The direct effect of H2O2 on Cl- secretion across intestinal epithelial cells has been previously examined using cultured T84 monolayers (27). In that study, H2O2 was shown to stimulate Cl- secretion in a concentration-dependent fashion. Furthermore, H2O2 significantly potentiated Cl- secretion in monolayers previously stimulated to secrete Cl- by cAMP-dependent secretagogues. Importantly, the augmented Cl- secretion under prestimulated conditions was only transient, and the level of total secretion dropped over a 30-min period to unstimulated levels. We have shown that pretreatment of T84 monolayers with H2O2 30 min before application of forskolin resulted in a concentration-dependent inhibition of Cl- secretion at concentrations between 500 µM and 5 mM H2O2. Moreover, both the stimulatory and inhibitory effects of H2O2 were blocked when we added catalase to the medium, thus indicating that H2O2 was specifically responsible for both effects.

Nguyen and Canada (27) speculated that the inhibitory effect of H2O2 was due to a decrease in Rt that they observed when monolayers were treated with >5 mM H2O2. At high concentrations, H2O2 was also shown to irreversibly decrease Rt in cultured renal epithelial cells (43). These changes have been attributed to the effect of H2O2 on the paracellular pathway. Importantly, we have shown that H2O2 inhibited forskolin-stimulated Cl- secretion at concentrations as low as 500 µM following a 30-min exposure and that inhibition was complete at 5 mM. Furthermore, in our study, 500 µM H2O2 did not significantly effect Rt. In addition, the release of LDH was not increased from monolayers treated with as much as 5 mM H2O2, compared with controls over the same period. Nguyen and Canada (27) also measured LDH release and failed to detect a significant increase at comparable concentrations following a 90-min exposure. In a study using the HT-29-18-Cl human colonic carcinoma cell line, cell survival decreased significantly at lower levels of H2O2 (100 µM) (41). However, the cells used in that study were not grown to confluence on permeable supports, which may have contributed to the cytotoxic effects of H2O2 reported. In addition, cell lines may have different sensitivities to H2O2 due to varying levels of catalase content. The fact that our results show that Rt and LDH release were not affected under basal conditions indicated that H2O2, at substantially lower levels than previously reported, significantly inhibited Cl- secretion across T84 monolayers through specific effects on the Cl- secretory pathway rather than through changes in the paracellular permeability of the monolayers.

In our study, bath H2O2 concentration decreased ~60% within 30 min following a bolus addition, suggesting that T84 cells consumed H2O2. When we treated T84 monolayers with aminotriazole, an inhibitor of endogenous catalase, the inhibitory effect of H2O2 was significantly potentiated. Similar effects of aminotriazole were also reported in HT-29-18-Cl cells subsequently treated with H2O2 (41). These findings indicate that endogenous catalase promotes the breakdown of H2O2 into water, thereby diminishing the inhibitory effects of H2O2 on Cl- secretion across the monolayers.

Although our data indicate that the inhibitory effects of H2O2 were not due to generalized cytotoxic effects, it was unclear which of the transport pathways involved in the secretory response were affected. When we permeabilized the basolateral membrane with nystatin and subsequently treated the monolayers with forskolin in the presence of an apical-to-basolateral Cl- gradient, a large increase in ICl resulted. Importantly, the magnitude of the ICl under these conditions was significantly reduced when cells were exposed to H2O2 before permeabilization, indicating an inhibitory effect of H2O2 on the apical membrane Cl- conductive pathway.

Reactive species, such as · OH and H2O2, have been previously shown to adversely affect ion channel function (25), possibly through the oxidation of key sulfhydryls and/or amino acid residues on the channel protein (23). Alternatively, the H2O2-induced decrease in ICl may be due to an inhibition of intracellular ATP levels. Previous work has shown that a fall in intracellular ATP concentration is one of the earliest changes observed during oxidative challenge (35). The role of ATP in regulating CFTR channel activity through a cAMP-dependent protein kinase (PKA)-mediated phosphorylation of the protein has been well established (42). Decreased intracellular ATP levels may alter cAMP production and metabolism and thus modify the regulatory pathway. More recently, however, a PKA-independent role for ATP-mediated regulation of CFTR channel activity has also been demonstrated (1, 4, 28). These observations led Bell and Quinton (4) to speculate that ATP binding to CFTR in T84 cells regulates apical membrane Cl- conductance in a manner that couples energy consumption by transport processes to ATP availability within the cell. They reported that the EC50 for ATP activation of CFTR was 3 mM. Consequently, the >40% decrease in ATP levels we observed following H2O2 treatment would be expected to inhibit the CFTR channel protein and therefore the apical membrane Cl- conductance. This conclusion is further supported by the fact that 5 mM 2-deoxyglucose, a concentration sufficient to decrease intracellular ATP levels by nearly 60%, mimicked the inhibitory effect of H2O2 on transepithelial ion transport.

Our data also demonstrate, in a definitive fashion, that H2O2 decreased Na+-K+-ATPase activity. Clinically, Na+-K+-ATPase activity was shown to be decreased by 75% in patients with ulcerative colitis compared with healthy individuals (29). Conclusions regarding the role reactive species play in modulating Na+-K+-ATPase function are presently mixed. Clerici et al. (6) reported that exposure of cultured alveolar type II cells to high concentrations of H2O2 (2.5 mM) for 20 min led to a 50% depletion of the Na+-dependent phosphate and alanine uptake and >92% depletion of cellular ATP levels. However, even for these very high concentrations of H2O2, the ouabain-sensitive 86Rb+ uptake of these cells was decreased by only 26%. Moreover, when these authors inhibited intracellular ATP levels with either antimycin or 2-deoxyglucose, ouabain-sensitive 86Rb+ uptake was unaffected. In contrast, Kim and Suh (20) reported that addition of H2O2 to the basolateral side of cultured alveolar type II cells mounted in Ussing chambers decreased the Isc with an IC50 of ~40 µM, which was an order of magnitude less than the corresponding apical IC50. Moreover, ouabain-sensitive 86Rb+ uptake was also inhibited to a higher degree by basolateral H2O2 administration than by apical H2O2 administration. These data led the authors to conclude that H2O2 directly inhibited the basolaterally located Na+-K+-ATPase.

Decreased Na+-K+-ATPase activity has been reported in both renal (18) and cardiac (21) cells after ischemia-reperfusion, a pathological condition known to result in increased production of reactive oxygen and nitrogen species, and after exposure of purified Na+-K+-ATPase to high concentrations of H2O2 and Fenton-type reagents. On the other hand, when xanthine and xanthine oxidase were used to generate superoxide, hydrogen peroxide, and hydroxyl radicals, the Isc across the ventral toad skin was decreased only when the reagents were applied to the apical side (25). This indicated that Na+-K+-ATPase was not damaged. Moreover, exposure of endothelial or P388D1 murine cells to 5 mM H2O2 did not alter their Na+-K+-ATPase activity, despite a large decrease in ATP levels (35).

In contrast to these studies, our finding that 2-deoxyglucose inhibited both intracellular ATP concentration and the INa indicates that ATP concentration and Na+-K+-ATPase activity are closely coupled in these cells. Furthermore, the results strongly suggest that the inhibitory effects of H2O2 on Na+-K+-ATPase activity in T84 monolayers likely result from its effects on intracellular ATP levels. The observed shift in the Hill coefficient suggests that H2O2 has increased the cooperativity between the Na+ binding sites. However, it is unclear whether this is related to decreased ATP levels or a direct effect of H2O2 on the protein.

Last, our results indicate that, in contrast to its effects on the apical membrane Cl- conductance and basolateral membrane Na+-K+-ATPase, H2O2 activated a basolateral membrane K+ conductance. Although the IK response was greatest in monolayers that were incubated with H2O2 for 30 min, it also increased ~50% within 5 min when the response was measured acutely. This may account for the transient increase in Cl- secretion observed immediately following H2O2 treatment. Outward IK across the basolateral membrane is required to counter the exit of Cl- across the apical membrane (8, 9). Under basal conditions, activation of basolateral membrane K+ channels hyperpolarizes the apical membrane, thereby increasing the driving force for Cl- efflux through apical membrane channels (1). The magnitude of the resulting Cl- secretory response was shown to be dependent on the level of Cl- channel activation when the basolateral K+ channels were activated (11, 46). The fact that we and others (27) have seen an increased stimulatory response to H2O2 in monolayers treated with cAMP-dependent secretagogues further supports the role of basolateral membrane K+ channels in the early H2O2 response.

H2O2 has been shown to directly activate K+ channels in neurons (33), pulmonary neuroendocrine cells (7, 40), carotid body chemoreceptor cells (39), and ventricular myocytes (17). In the last instance, the sensitivity of the channel to H2O2 was increased fivefold in the presence of 10 µM ADP. Although there is currently no evidence of H2O2-induced K+ channel activation in epithelial cells, K+ channels that are activated by increasing ADP levels are present in enterocytes (26, 32). It is currently unknown whether such a channel exists in T84 cells and thus what role it may play in the observed responses to H2O2. An alternative mechanism to explain the H2O2-induced basolateral K+ channel activation in T84 cells is through a Ca2+-mediated pathway. H2O2 is known to elevate intracellular Ca2+, and many of the effects of H2O2 are, at least in part, attributed to elevations in intracellular Ca2+ concentration (14). Moreover, Ca2+ has been shown to participate in agonist-induced Cl- secretion in T84 cells by activating basolateral membrane K+ channels (12, 24, 46). Our results indicate that H2O2 increased the Ba2+-sensitive IK. However, there is disagreement in the literature as to whether or not Ca2+ activates a Ba2+-sensitive K+ channel in T84 cells. In early work on T84 cells, Mandel et al. (24) demonstrated that Ba2+ blocked the Isc transient and the basolateral K+ efflux produced by the Ca2+ ionophore A-23187 in intact T84 monolayers. This contrasts with later studies (10, 31, 46) that failed to show an effect of Ba2+ on K+ channel activated by carbachol or the ionophore ionomycin. It is unclear to what these discrepancies are due; however, it should be noted that the later studies were not conducted on intact monolayers and that perturbations to the system may have altered the responses. It is important to note that when we treated monolayers with BAPTA-AM, both the transient increase in Isc and inhibition of the forskolin-induced response by H2O2 were abolished. This indicates that the effects of H2O2 are mediated by Ca2+, although the precise mechanisms are presently unknown.

In summary, the H2O2-induced modulation of Cl- secretion across T84 cell monolayers is multifaceted. H2O2 inhibits both the apical membrane Cl- conductive pathway and basolateral membrane Na+-K+-ATPase. Either of these effects would be expected to inhibit transepithelial Cl- secretion. Inhibition of these transport processes appears to be dependent on intracellular Ca2+ and linked to decreased intracellular ATP levels, which may explain the relatively long onset of the inhibitory effect. Acutely, H2O2 activates a basolateral K+ conductance that may account for the transient increase in Cl- secretion observed immediately following H2O2 treatment. In conclusion, these results indicate that the principal effect of H2O2 on colonic Cl- secretion is that of inhibition. Reduced fluid secretion, secondary to reduced Cl- secretion, would compromise the mucosal hygiene role of the crypt cells within the colonic epithelium. This would be expected to result in an inefficient removal of pathogens and noxious substances from the mucosa, thereby exacerbating their untoward effects.

    ACKNOWLEDGEMENTS

We thank Dr. Lan Chen and Tanta Myles for technical assistance.

    FOOTNOTES

Y. Guo was partially supported by National Institutes of Health (NIH) Training Grant HL-07553-T32. Additionally, this project was supported by NIH Grants DK-01935 (to M. D. DuVall), HL-51173 (to S. Matalon), and HL-31197 (to S. Matalon) and Office of Naval Research Grant 000014-93-0785 (to S. Matalon).

M. D. DuVall is a Parker B. Francis Fellow in Pulmonary Research.

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

Address for reprint requests: S. Matalon, Dept. of Anesthesiology, University of Alabama, 619 19th St., THT 940, Birmingham, AL 35233.

Received 15 April 1998; accepted in final form 31 July 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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