Hydrogen Peroxide Stimulates the Epithelial Sodium Channel through a Phosphatidylinositide 3-Kinase-dependent Pathway

Abstract

Recent studies indicate that oxidative stress mediates salt-sensitive hypertension. To test the hypothesis that the renal epithelial sodium channel (ENaC) is a target of oxidative stress, patch clamp techniques were used to determine whether ENaC in A6 distal nephron cells is regulated by hydrogen peroxide (H₂O₂). In the cell-attached configuration, H₂O₂ significantly increased ENaC open probability (P_o) and single-channel current amplitude but not the unit conductance. High concentrations of exogenous H₂O₂ are required to elevate intracellular H₂O₂, probably because catalase, the enzyme that promotes the decomposition of H₂O₂ to H₂O and O₂, is highly expressed in A6 cells. The effect of H₂O₂ on ENaC P_o was enhanced by 3-aminotriazole, a catalase inhibitor, and abolished by overexpression of catalase, indicating that intracellular H₂O₂ levels are critical to produce the effect. However, H₂O₂ did not directly activate ENaC in inside-out patches. The effects of H₂O₂ on ENaC P_o and amiloride-sensitive Na⁺ current were abolished by inhibition of phosphatidylinositide 3-kinase (PI3K). Confocal microscopy data showed that H₂O₂ elevated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃) in the apical membrane by stimulating PI3K. Because ENaC is stimulated by PI(3,4,5)P₃, these data suggest that H₂O₂ stimulates ENaC via PI3K-mediated increases in apical PI(3,4,5)P₃.

Introduction

ENaC² in the distal nephron plays an important role in maintaining Na⁺ homeostasis and consequently controls systemic blood pressure. Excess sodium reabsorption due to enhanced ENaC activity can lead to hypertension, as is seen in Liddle syndrome (1, 2) and other genetic diseases (3). Surprisingly, whether high salt intake induces hypertension by targeting ENaC has not been examined. High salt intake can elevate both superoxide (O₂^⨪) (4, 5) and H₂O₂ (6) in the kidney by stimulating NAD(P)H oxidase (7–9), and O₂^⨪ can increase aldosterone-mediated stimulation of ENaC by scavenging nitric oxide (NO) (10). However, it is still unknown whether H₂O₂, a product of O₂^⨪, also stimulates ENaC.

H₂O₂ may be more important than other reactive oxygen species (ROS) in the pathogenesis of salt-sensitive hypertension. There are two lines of evidence that support a role for H₂O₂ in salt-sensitive hypertension. First, transgenic mice overexpressing the human catalase gene are found to be less sensitive to hypertension-inducing agents, such as norepinephrine and angiotension II (11). Second, among ROS, H₂O₂ is a relatively stable and mild oxidant, suggesting that it may have a longer effect than O₂^⨪. In this context, H₂O₂ acts as an intracellular messenger that modulates the function of several types of transmembrane proteins (12), including ATP-sensitive potassium (K_ATP) channels (13). Intracellular H₂O₂ concentrations are balanced between the rate of production determined by NAD(P)H oxidase-mediated production of O₂^⨪ and the rate of degradation determined by catalase and glutathione peroxidase (14).

ROS, including H₂O₂, stimulate the non-receptor tyrosine kinase, c-Src (15). c-Src is known to be directly associated with p85, the regulatory subunit of PI3K (16). Tyrosine phosphorylation of p85 reduces its inhibitory effect on PI3K (17). These studies indicate that ROS may stimulate PI3K and consequently promote the synthesis of PI(3,4,5)P₃. We (18–20) and others (21, 22) have previously shown that PI(3,4,5)P₃ stimulates ENaC. Taken together, these studies suggest that H₂O₂ may stimulate ENaC by elevating PI(3,4,5)P₃ through activation of PI3K. In the present study, using patch clamp techniques combined with confocal microscopy analysis of apical PI(3,4,5)P₃ levels, we show that H₂O₂ stimulates ENaC in A6 distal nephron cells via PI3K-dependent elevation of PI(3,4,5)P₃ in the apical membrane.

MATERIALS AND METHODS

Cell Culture

Xenopus A6 distal nephron cells were purchased from the American Type Culture Collection (Manassas, VA). Under previously used culture conditions, basal activity of ENaC in A6 cells is relatively high (23). It is difficult to see and analyze any stimulatory effect when basal ENaC activity is high. Therefore, in the present study, we modified the culture medium to reduce basal ENaC activity. The medium contained 2 parts of DMEM/F-12 (1:1) medium (Invitrogen), 1 part of H₂O, 15 mm NaHCO₃ (total Na⁺ = 101 mm, which is ideal for amphibian A6 cells), 2 mm l-glutamine, 10% fetal bovine serum (Invitrogen), 25 units/ml penicillin, 25 units/ml streptomycin. A6 cells were cultured in plastic flasks in the presence of 1 μm aldosterone at 26 °C and 4% CO₂. After the cells became 70% confluent in plastic flasks, A6 cells were plated on the polyester membrane of either Snapwell inserts (Corning Costar Co.) for patch clamp experiments) or Transwell inserts (Corning Costar Co.) for confocal microscopy experiments and measurement of amiloride-sensitive Na⁺ current). The cells were cultured for 3–4 weeks to allow them to be fully polarized before each experiment.

Patch Clamp Recordings

Both cell-attached and inside-out recordings of ENaC single-channel currents from A6 distal nephron cells were carried out using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). As described previously (24), prior to the experiments, A6 cells cultured on the polyester membrane of Snapwell inserts were thoroughly washed with NaCl solution containing 100 mm NaCl, 3.4 mm KCl, 1 mm CaCl₂, 1 mm MgCl₂, and 10 mm HEPES; pH was adjusted to 7.4 with NaOH. The glass micropipette was filled with NaCl solution (the pipette resistance is 7–10 megaohms). In cell-attached experiments, NaCl solution was used for both the luminal and basolateral bath. For experiments using the inside-out patch clamp configuration, KCl solution was used for the cytoplasmic bath, which contained 100 mm KCl, 5 mm NaCl, 1 mm MgCl₂, and 10 mm HEPES (50 nm free Ca²⁺ after titration with 1 mm EGTA); pH was adjusted to 7.2 with KOH. Single-channel currents were obtained with applied pipette potentials of either 0 mV for cell-attached recordings or +60 mV for inside-out recordings, filtered at 1 kHz, and sampled every 50 μs with Clampex 8.0 software. All experiments were conducted at room temperature. The total numbers of functional channels in the patch were estimated by observing the number of peaks detected on the current amplitude histograms during an at least 20-min recording period including the period after application of H₂O₂. Each experiment was started after the first 2-min recordings when the ENaC activity had stabilized. The open probability (P_o) of ENaC before (−3–0 min) and after each experimental manipulation (every 3-min period) was calculated using Clampfit 9.0.

Measurement of Amiloride-sensitive Na⁺ Current

After they were cultured on the polyester membrane of Transwell inserts for 3–4 weeks, A6 cells were fully polarized and formed a cell monolayer. The voltage (V) and resistance (R) across the cell monolayer were measured with electrodes connected to a volt ohm meter, the EVOM (World Precision Instruments, Sarasota, FL). The transepithelial current (I) was calculated according to Ohm's law (I = V/R) and corrected for the surface area of the polyester membrane. Because H₂O₂ could alter the membrane potential by stimulating iberiotoxin-sensitive K⁺ channels (data not shown), we used the amiloride-sensitive Na⁺ current (I_Na) rather than the transepithelial current to analyze ENaC activity. To calculate the amiloride-sensitive I_Na, the total transepithelial current was subtracted with the current after blockade of ENaC with 1 μm amiloride.

Transfection of A6 Cells with Enhanced Green Fluorescence Protein-tagged Pleckstrin Homology Domain of Akt (EGFP-PH-Akt)

In this set of experiments, A6 cells were plated on the polyester membrane of Transwell inserts at a high density to allow the cells to be confluent within 3 days. To facilitate transfection of confluent A6 cells, we treated confluent A6 cells with Ca²⁺-free and Mg²⁺-free PBS (Invitrogen). The PBS was modified with H₂O (3 parts of PBS with 1 part of H₂O) to match the osmolarity of amphibian cells. After treatment with the modified PBS for 20 min (to break the tight junctions), cells were incubated with transfection reagent containing EGFP-PH-Akt DNA construct and Lipofectamin 2000 for 6 h and then incubated with regular culture medium for 2 days before performing the confocal microscopy experiments.

Confocal Microscopy Imaging

To detect PI(3,4,5)P₃ levels in A6 cells, A6 cells were transiently transfected with EGFP-PH-Akt. To detect the levels of intracellular ROS, A6 cells were stained with a membrane-permeable, ROS-sensitive, fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate. Prior to the confocal miscroscopy experiments, cells were washed twice with NaCl solution. Immediately following each experimental manipulation, the polyester membrane that supports the A6 cell monolayer was quickly excised and mounted on a glass slide with a drop of NaCl solution to keep the cells alive. Confocal microscopy xy or xz scanning of A6 cells was accomplished within 5 min. xy optical sections were performed to provide a flat view of the cells near the apical membrane, across the lateral membrane, or near the basal membrane. xz optical sections were also performed to provide a lateral view of the cells. In each set of experiments, images were taken using the same parameter settings.

Measurement of Intracellular H₂O₂

It has been suggested that aquaporins are required for H₂O₂ to move across the cell membrane (25). After the cells were challenged with exogenous H₂O₂, the levels of intracellular H₂O₂ remained unknown. Therefore, H₂O₂ concentrations in A6 cells after treatment with exogenous H₂O₂ were measured. After treatment with H₂O₂ at different concentrations for 10 min, cells were washed five times in ice-cold NaCl solution. Cells were then bathed in calcium- and magnesium-free PBS for 15 min and treated with trypsin for 1–2 min. Cells were collected by centrifuging at 600 × g for 5 min, washed twice with NaCl solution, and then suspended in 350 μl of NaCl solution. H₂O₂ concentrations in live A6 cells were measured using the Amplite fluorimetric hydrogen peroxide assay kit (ABD Bioquest, Sunnyvale, CA) according to the manufacturer's directions. Briefly, a 50-μl cell suspension was incubated with an equal volume mixture of Amplite Red peroxidase substrate and horseradish peroxidase for 30 min at room temperature. The signal at 570 nm in a 96-well microtiter plate was detected and recorded by a microplate reader. Cell number was counted and used to calculate intracellular H₂O₂ according to the standard curve.

Western Blotting

A6 cells were cultured as described above. Cell lysates (100 μg) were loaded and electrophoresed on 10% SDS-polyacrylamide gels for 60–90 min. Gels were blotted onto polyvinylidene fluoride (PVDF) membranes for 1.5 h at 50 V. After 1 h of blocking with 5% BSA-PBST buffer, PVDF membranes were incubated with primary antibody (1:10,000 dilution) of rabbit monoclonal antibody to catalase (Abcam; ab76024) overnight at 4 °C and then incubated with horseradish peroxidase (HRP)-conjugated sheep anti-rabbit IgG secondary antibody (1:10,000 dilution; GE Healthcare) for 1 h after four vigorous washes. Finally, blots were visualized with chemiluminescence using the ECL Plus Western blotting detection system (GE Healthcare).

Chemicals

Most chemicals were purchased from Sigma-Aldrich. H₂O₂ was purchased from Fisher. H₂O₂ was diluted with NaCl solution before it was added to either luminal or basolateral bath. All of the solutions were either premade and stored in a −20 °C freezer or freshly made before the experiments. All of the concentrations listed in this study represent the final concentrations.

Statistical Analysis

Data are reported as mean values ± S.E. Statistical analysis was performed with SigmaPlot and SigmaStat software (Jandel Scientific). Paired t test was used for comparisons between pre- and post-treatment activities. Analysis of variance was used for multiple comparisons among various treatment groups or at different time points of one treatment group. Results were considered significant if p was <0.01.

RESULTS

Basolateral H₂O₂ Stimulates ENaC in Cell-attached Patches

Recent studies suggest that elevation of H₂O₂ in the kidney participates in salt-sensitive hypertension (6, 26), implying that the activity of sodium transporters might be increased by H₂O₂. In the present study, cell-attached patch clamp experiments were performed to determine whether H₂O₂ would stimulate ENaC. Because ENaC in A6 distal nephron cells is well characterized, we used the A6 cell line as a model for sodium-transporting epithelial cells. We intended to determine the role of intracellular rather than extracellular H₂O₂ in regulating ENaC. Therefore, H₂O₂ was applied outside the patch pipette to the cells rather than to inside the pipette with the expectation that high concentrations of extracellular H₂O₂ would drive H₂O₂ to move across the cell membrane and increase intracellular H₂O₂. Our results show that the addition of 3 mm H₂O₂ to the basolateral bath significantly increased both ENaC open probability (P_o) and single-channel current amplitude with a latency of about 12 min (Fig. 1). In contrast, the addition of saline (NaCl solution, used for making up H₂O₂ stock solution) to the basolateral bath did not affect ENaC activity. These data show that H₂O₂ stimulates ENaC from the basolateral side in distal nephron cells but with a long latency.

FIGURE 1.

Basolateral H₂O₂ elevates ENaC open probability (P_o) and single-channel current amplitude in A6 distal nephron cells. A, a representative single-channel record from a cell-attached patch before and after the addition of 3 mm H₂O₂ to the basolateral bath (inset panel a shows increases in single-channel current amplitude after H₂O₂). B, a representative single-channel record from a cell-attached patch before and after the addition of saline (used both for the bath and for making up H₂O₂ stock solution) to the basolateral bath (inset panel b shows no change in single-channel current amplitude after saline). I and II, zoomed-in regions. Downward events represent channel openings. C, summary plots of ENaC (P_o) before and after the addition of either H₂O₂ (filled circles) or saline (open circles) to the basolateral bath. Each circle represents mean P_o calculated from 3-min single-channel records in A6 cells. Error bars, S.E.

Luminal H₂O₂ Also Stimulates ENaC in Cell-attached Patches

In the above experiments, H₂O₂ was applied to the basolateral bath. If H₂O₂ stimulates ENaC by causing intracellular oxidative stress, it must pass through the basolateral membrane in order to reach the apical membrane where ENaC is located. Therefore, we examined the effects of H₂O₂ applied in the luminal bath on ENaC. Compared with basolateral H₂O₂, luminal H₂O₂ at the same concentration of 3 mm also significantly increased both ENaC P_o and single-channel current amplitude but with a shorter latency of about 6 min (Fig. 2, A, C, and D). The current-voltage relationship showed that H₂O₂ did not alter ENaC unit conductance (Fig. 2E). In contrast, the addition of 0.3 mm H₂O₂ to the luminal bath had no effect on ENaC P_o and only slightly increased ENaC single-channel current amplitude (Fig. 2, B, C, and D).

FIGURE 2.

Luminal H₂O₂ also elevates ENaC P_o and single-channel current amplitude in A6 distal nephron cells. A and B, representative single-channel records from cell-attached patches in A6 cells before and after the addition of either 3 mm or 0.3 mm H₂O₂ to the luminal bath. C, summary plots of ENaC P_o before and after the addition of either 3 mm (filled circles) or 0.3 mm H₂O₂ (open circles) to the luminal bath. D, summary plots of single-channel current amplitude before and after H₂O₂. E, the current-voltage (I-V) relationship before and after 3 mm H₂O₂. Error bars, S.E.

A High Concentration of Exogenous H₂O₂ Is Required to Cause Intracellular Oxidative Stress

It has been reported that high concentrations of H₂O₂ are required to produce significant acute effects in some types of cells (27, 28). However, it remains unknown whether high concentrations of extracellular H₂O₂ are necessary to elevate intracellular H₂O₂ and produce significant oxidative stress. Therefore, the relationship between exogenous H₂O₂ load and intracellular oxidative stress was determined. The data show that exogenous H₂O₂ does not significantly elevate intracellular ROS until the concentration of exogenous H₂O₂ was in the millimolar range (Fig. 3A). To determine whether the oxidative stress is due to elevation of intracellular H₂O₂, intracellular H₂O₂ was measured using the Amplite^TM hydrogen peroxide assay kit. Luminal exposure of A6 cells to relatively high concentrations of H₂O₂ does increase intracellular H₂O₂ from 3.5 ± 0.2 (control; n = 9) to 5.1 ± 0.4 nm (0.3 mm H₂O₂ for 10 min; n = 9; p < 0.05) or 12.2 ± 0.7 nm (3 mm H₂O₂ for 10 min; n = 9; p < 0.001) (Fig. 3B). This small but significant change suggests that either the permeability of the cell membranes to H₂O₂ is low or the rate of H₂O₂ degradation is high. Because the level of intracellular H₂O₂ is tightly controlled by catalase, we performed Western blot experiments and found that catalase is highly expressed in A6 cells (Fig. 3B, inset). These data suggest that a high concentration of exogenous H₂O₂ is required to elevate intracellular H₂O₂ because catalase rapidly breaks down H₂O₂ that entered the cells.

FIGURE 3.

Exogenous H₂O₂ causes intracellular oxidative stress. A shows that luminal H₂O₂ at 3 mm, but not at 0.3 mm, significantly elevated intracellular ROS. Confluent A6 cells were stained with 2′,7′-dichlorodihydrofluorescein diacetate, a membrane-permeable fluorescent probe detecting intracellular ROS. These confocal microscopy data represent consistent results from three experiments. B shows that the elevation was due to increases in intracellular H₂O₂ in A6 cells. Intracellular H₂O₂ was determined using the Amplite^TM hydrogen peroxide assay kit (ABD Bioquest, Sunnyvale, CA). The inset shows Western blot of catalase in A6 cells. The cells were under control conditions (left) or treated with either 0.3 mm (middle) or 3 mm H₂O₂ (right). Error bars, S.E.

Inhibition of Catalase Facilitates H₂O₂ Stimulation of ENaC

If exogenously added H₂O₂ stimulates ENaC by elevating intracellular H₂O₂, but the intracellular concentration of H₂O₂ is kept low by the action of catalase, then inhibition of catalase should facilitate the effect of exogenous H₂O₂ on ENaC activity. To test this hypothesis, A6 cells were exposed to 1 mm H₂O₂ in the absence (control) and presence of 20 mm 3-aminotriazole, a catalase inhibitor. Under control conditions, 1 mm H₂O₂ tended to elevate ENaC P_o but not in a statistically significant manner. In contrast, after inhibition of catalase with 3-aminotriazole, the effect of 1 mm H₂O₂ on ENaC P_o became statistically significant (Fig. 4). Inhibition of catalase did not alter the effect of 1 mm H₂O₂ on ENaC single-channel current amplitude, indicating that H₂O₂ may elevate ENaC current amplitude through another pathway that is very sensitive and already saturated by 1 mm H₂O₂ alone. 3-Aminotriazole alone did not acutely affect the basal activity of ENaC in A6 cells. This lack of effect is probably due to low basal H₂O₂ production when A6 cells were in the patch chamber and bathed with glucose-free saline at room temperature. These data suggest that H₂O₂ regulates ENaC via an intracellular mechanism.

FIGURE 4.

Inhibition of catalase enhances the effect of H₂O₂ on ENaC P_o. A, representative single-channel record from a cell-attached patch before and after the addition of 1 mm H₂O₂ to the luminal bath. B, representative single-channel record from a cell-attached patch before and after the addition of 1 mm H₂O₂ to the luminal bath in the presence of 20 mm 3-aminotriazole, a catalase inhibitor. C, representative single-channel record from a cell-attached patch before and after the addition of 20 mm 3-aminotriazole. D, summary plots of ENaC P_o before and after the addition of 1 mm H₂O₂ alone (open circles), 1 mm H₂O₂ in the presence of a catalase inhibitor (20 mm 3-aminotriazole; filled triangles) or the catalase inhibitor alone (20 mm 3-aminotriazole; open triangles) to the luminal bath. E, summary plots of single-channel current amplitude before and after H₂O₂ in the absence (left bars) or presence (middle bars) of the catalase inhibitor. The catalase inhibitor alone served as another control (right bars). Error bars, S.E.

Overexpression of Catalase Abolishes H₂O₂ Stimulation of ENaC

The data described above support the hypothesis that highly expressed catalase may account for the resistance of A6 cells to H₂O₂. To determine whether exogenous H₂O₂ stimulates ENaC by elevating intracellular H₂O₂, we overexpressed catalase in A6 cells. To monitor the expression of this enzyme, we subcloned mitochondrial catalase into pEYFP-C1 vector to form the pEYFP-C1-mito-Cat construct. A6 cells were then transiently transfected with this construct. The cells expressing this exogenous catalase were identified by yellow fluorescence. In an A6 cell that was under transfecting conditions but did not express this catalase, the addition of 3 mm H₂O₂ to the luminal bath elevated both ENaC P_o and single-channel current amplitude (Fig. 5A) but failed to produce an effect on ENaC in an A6 cell that expressed the exogenous catalase (the cell with the fluorescence; Fig. 5B). These results suggest that the effect of exogenous H₂O₂ on ENaC activity is highly dependent on the levels of intracellular H₂O₂.

FIGURE 5.

Overexpression of a catalase abolishes H₂O₂ stimulation of ENaC. A and B, representative single-channel records from cell-attached patches in A6 cells either under control conditions (A) or when overexpressing a catalase (B) before and after the addition of 3 mm H₂O₂ to the luminal bath. C, summary plots of ENaC P_o before and after 3 mm H₂O₂ in A6 cells either under control conditions (filled circles) or when overexpressing a catalase (open circles). D, moderate increases in single-channel current amplitude after the addition of 3 mm H₂O₂ in control A6 cells but not in the cells overexpressing a catalase. Error bars, S.E.

H₂O₂ Does Not Affect ENaC Activity in Inside-out Patches

Previously reported studies suggested that H₂O₂ stimulates voltage-gated K⁺ channels of the Kv7 family by directly oxidizing cysteine residues of this channel protein (29). Therefore, we originally hypothesized that H₂O₂ might stimulate ENaC by oxidizing intracellular cysteine residues of ENaC. However, using the inside-out configuration, the cytosolic surface of ENaC was exposed to either 1 or 3 mm H₂O₂. We found that H₂O₂ did not alter either ENaC P_O (Fig. 6, A and C) or ENaC single-channel current amplitude (Fig. 6C). These data suggest that H₂O₂ stimulates ENaC probably not by directly oxidizing ENaC but rather via activation of signal transduction pathways.

FIGURE 6.

Exposure of the cytoplasmic side of ENaC to H₂O₂ does not affect its activity. A, a representative inside-out patch showing that ENaC activity was unchanged after the addition of 1 mm or 3 mm H₂O₂ to the “cytoplasmic” bath. B, summary plots of ENaC P_o before and after the addition of 1 mm (filled circles) or 3 mm H₂O₂ (open circles) to the “cytoplasmic” bath. C, no change in single-channel current amplitude after the addition of 1 or 3 mm H₂O₂. +60 mV was applied to the patch pipette. Error bars, S.E.

Inhibition of PI3K Abolishes the Effect of H₂O₂ on ENaC P_O

To determine whether PI3K mediates the effects of H₂O₂ on ENaC activity, prior to the addition of 3 mm H₂O₂ to the luminal bath, A6 cells were pretreated with 5 μm LY294002, a PI3K inhibitor, for 5 min. Previous studies suggested that PI(3,4,5)P₃ is produced in the inner leaflet of the basolateral membrane and can freely diffuse in the inner leaflet to reach the apical membrane (30). Therefore, the PI3K inhibitor was added to the basolateral bath. We found that after the pretreatment, H₂O₂ no longer elevated ENaC P_o but still increased ENaC single-channel current amplitude (Fig. 7, A–D). In contrast, LY294002 alone only slightly reduced ENaC P_o when basal ENaC P_o was low and had no effect on ENaC single-channel current amplitude. These data suggest that the effect of H₂O₂ on ENaC P_o, but not on ENaC single-channel current amplitude, is mediated by a PI3K-dependent pathway.

FIGURE 7.

Inhibition of PI3K abolishes the effect of H₂O₂ on ENaC P_o. A, a representative single-channel record from a cell-attached patch before and after the addition of 3 mm H₂O₂ to the luminal bath in the presence of 5 μm LY294002 (added to the basolateral bath), a PI3K inhibitor. B, a representative single-channel record from a cell-attached patch before and after the addition of 5 μm LY294002 to the basolateral bath. C, summary plots of ENaC P_o before and after the addition of either 3 mm H₂O₂ to the luminal bath in the presence of 5 μm LY294002 (filled circles) or 5 μm LY294002 alone to the basolateral bath (open circles). D, summary plots of single-channel current amplitude before and after either 3 mm H₂O₂ in the presence of 5 μm LY294002 (left bars) or 5 μm LY294002 alone (right bars). E, summary plots of amiloride-sensitive I_Na. A6 cells were in control conditions, apically treated with 3 mm H₂O₂ for 15 min, basolaterally treated with 5 μm LY294002 for 15 min, or basolaterally pretreated with 5 μm LY294002 for 5 min followed by apical treatment with 3 mm H₂O₂ for 15 min. Error bars, S.E.

Inhibition of PI3K Abolishes the Effect of H₂O₂ on Amiloride-sensitive Na⁺ Current

To confirm the results from single-channel recordings, amiloride-sensitive Na⁺ current (I_Na) was measured. As shown in Fig. 7E, under control conditions when A6 cells were cultured with our modified culture medium, base-line I_Na was 2.1 ± 0.1 μA/cm² (n = 10). After the A6 cell monolayer was apically treated with 3 mm H₂O₂ for 15 min, I_Na was increased to 6.5 ± 0.2 μA/cm² (n = 10, p < 0.001). However, H₂O₂ failed to increase I_Na (2.3 ± 0.1 μA/cm², n = 10) in A6 cells pretreated with 5 μm LY294002 for 5 min. Treatment of the cells with 5 μm LY294002 alone for 15 min did not alter I_Na, probably because PI3K is inactive in A6 cells under basal conditions. These results are consistent with the data from patch clamp single-channel recordings.

H₂O₂ Induces PI3K-dependent Elevation of PI(3,4,5)P₃ in the Apical Membrane of A6 Cells

Because our previous studies have shown that PI(3,4,5)P₃, one of the PI3K products, stimulates ENaC (18–20), H₂O₂ may stimulate ENaC via a PI3K-dependent elevation of PI(3,4,5)P₃ in the apical membrane. To test this hypothesis, a previously established method (31) was used to track PI(3,4,5)P₃ in living A6 cells. The cells were transiently transfected with the EGFP-PH-Akt construct in which the PH domain could selectively bind to PI(3,4,5)P₃. Consistent with previous studies (31), the data showed that under basal conditions, PI(3,4,5)P₃ was undetectable in the apical membrane of A6 cells but was mainly located in the lateral and basal membranes. Treatment of the cells with 3 mm H₂O₂ for 15 min induced the appearance of PI(3,4,5)P₃ in the apical membrane. In contrast, when the cells were pretreated with 5 μm LY294002 for 5 min to inhibit PI3K, H₂O₂ failed to induce apical PI(3,4,5)P₃ production (Fig. 8). Because our patch clamp data showed that inhibition of PI3K abolished the effect of H₂O₂ on ENaC P_o, these confocal microscopy data suggest that apical PI(3,4,5)P₃ production mediates H₂O₂ stimulation of ENaC.

FIGURE 8.

H₂O₂ induces PI3K-dependent elevation of PI(3,4,5)P₃ in the apical membrane of A6 cells. Left images show confocal microscopy XY sections of apical, lateral, and basal membranes of A6 cells. Right images show XZ sections A6 cells. The cells were transiently transfected with GFP-Akt-PHD to monitor PI(3,4,5)P₃ localization. The experiments were performed either under control conditions (A) or when the cells were treated with 3 mm H₂O₂ for 15 min in the absence (B) or the presence of 5 μm LY294002 (added to the basolateral bath) (C). The data represent three sets of experiments showing similar results. Error bars, S.E.

DISCUSSION

The present study shows that H₂O₂ increases both ENaC activity and apical PI(3,4,5)P₃ via a PI3K-dependent pathway. Because we previously showed that PI(3,4,5)P₃ stimulates ENaC in A6 cells (18–20), H₂O₂ probably stimulates ENaC by causing a PI3K-dependent elevation of PI(3,4,5)P₃ in the apical membrane. However, PI(3,4,5)P₃ should first be produced in the inner leaflet of the basolateral membrane and then diffused in the inner leaflet to the apical membrane because the PI3K inhibitor LY294002 was added to the basolateral bath. This hypothesis is supported by previous studies, which showed that PI(3,4,5)P₃ can laterally diffuse in the inner leaflet of epithelial cell membranes from the basolateral membrane domain, where it is generated, to the apical membrane domain (30). Theoretically, the apical PI(3,4,5)P₃ would be rapidly degraded because the lipid phosphatase PTEN (phosphatase and tensin homolog) is located in the apical membrane (31). However, a sustained elevation of apical PI(3,4,5)P₃ was observed after treatment of the cells with H₂O₂. This is not surprising because H₂O₂ not only activates PI3K but also inactivates PTEN by oxidizing its cysteine residues (32). Because the cytosolic domains of ENaC also contain cysteine residues, we reasoned that H₂O₂ may stimulate ENaC by oxidizing these residues. However, results from experiments using the inside-out or excised patch configuration show that H₂O₂ is unable to affect ENaC activity. The results suggest that H₂O₂ does not directly regulate ENaC but rather alters ENaC activity via a PI3K-dependent pathway.

In the present study, exogenous H₂O₂ was used as a tool to manipulate the levels of intracellular H₂O₂. In A6 cells, a high exogenous concentration of H₂O₂ is required to counteract the efficient catalase present in these cells, elevate intracellular H₂O₂, and regulate ENaC. However, H₂O₂, even at 10 mm, did not result in either cell lysis or apoptosis of A6 cells (supplemental Fig. 1). Based on these data, the concentrations of H₂O₂ used for the current study (up to 3 mm) should not result in any nonspecific effects due to cellular damage. Therefore, such high concentrations of H₂O₂ can be used as a tool to manipulate the levels of intracellular H₂O₂ in A6 cells. This argument is probably also true for neurons because in the hippocampus, the IC₅₀ for extracellular H₂O₂ to affect postsynaptic potentials is nearly 6 mm (33). Because, in the present study, H₂O₂ was applied outside the patch pipette (not to ENaC in the patch), it remains to be determined whether extracellular H₂O₂ could also directly regulate ENaC by oxidizing cysteine and/or histidine residues in the extracellular loops of ENaC subunits.

Compared with previously published studies from us and others (23, 34), ENaC mean P_o presented in this study is relatively low. In previous reports, ENaC mean P_o was calculated according to the number of active channels in the patch estimated under resting conditions. This estimation excluded any silent channels present in the patch. The mean P_o reflects only the value of active channels. In the present study, we found that the number of active channels was elevated after treatment with H₂O₂. This elevation is due to activation of silent channels rather than insertion of new channels in the patch because previous work also with A6 cells suggests that insertion of new channels never occurred in a patch that had already been formed (35). Therefore, the elevated number of channels after H₂O₂ was used to calculate base-line ENaC P_o. This P_o reflects the mean P_o value of both active and silent channels. Therefore, the P_o value reported here is much lower than those published previously. However, the open time of each active channel is still several s (see the zoom-in single-channel traces in each figure).

In order to clearly see the stimulatory effect of H₂O₂ on ENaC activity, we modified culture medium to reduce basal ENaC activity (see “Methods and Materials”). Compared with the culture medium we used previously (23), the modified culture medium contained less NaHCO₃ (15 mm versus 25 mm). Because recent studies have shown that HCO₃⁻ stimulates ENaC (36), less HCO₃⁻ in culture medium may account, at least in part, for low basal ENaC activity observed in the present study. We noticed that inhibition of PI3K did not reduce basal ENaC P_o and amiloride-sensitive Na⁺ current. Because our confocal microscopy data showed that PI(3,4,5)P₃ was undetectable in the apical membrane of A6 cells under basal conditions, we argue that lack of PI(3,4,5)P₃ might account for both the low basal ENaC activity and the insensitivity to the PI3K inhibitor, LY294002. However, it remains unknown whether our modified culture conditions could affect the levels of apical PI(3,4,5)P₃.

Our data showed that H₂O₂ increased ENaC single-channel current amplitude. Because H₂O₂ did not alter ENaC single-channel conductance, the increased current amplitude is probably caused by elevation of the driving force for Na⁺ influx. First, the electrical driving force (the membrane potential) may be elevated because H₂O₂ stimulates both K_ATP channels (13) and large conductance, calcium- and voltage-activated potassium (BK) channels (37). Second, the chemical driving force (Na⁺ concentration gradient) may also be elevated because H₂O₂ also stimulates Na⁺,K⁺-ATPase (38), which could decrease intracellular Na⁺. Investigation of the underlying mechanism will be the direction of our future studies. We noticed that the effect of H₂O₂ on single-channel amplitude in the control experiment shown in Fig. 5 was less significant than those observed in any other experiments. Because fully polarized epithelial cells are difficult to transfect, poorly polarized cells were used in the experiments shown in Fig. 5. In addition, prior to the transfection, the cells were serum-deprived for 24 h. Therefore, we argue that poor polarization and serum deprivation may minimize the effect of H₂O₂ on ENaC single-channel current amplitude, presumably by reducing functional K_ATP, BK, and/or Na⁺,K⁺-ATPase in the plasma membrane.

In summary, the present study provides the first evidence that H₂O₂ not only enhances ENaC P_o by elevating PI(3,4,5)P₃ in the apical membrane via activation of PI3K but also increases ENaC single-channel current amplitude via a PI3K-independent pathway. Because H₂O₂ in the kidney is elevated in salt-sensitive rats on a high salt diet (6), our in vitro data presented here imply a possible role of ENaC in salt-sensitive hypertension.