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. 2015 May 1;308(9):L943-52.
doi: 10.1152/ajplung.00213.2014. Epub 2015 Feb 20.

Oxidized glutathione (GSSG) inhibits epithelial sodium channel activity in primary alveolar epithelial cells

Charles A Downs  1 , Lisa Kreiner  2 , Xing-Ming Zhao  3 , Phi Trac  2 , Nicholle M Johnson  2 , Jason M Hansen  4 , Lou Ann Brown  4 , My N Helms  5
Affiliations

Oxidized glutathione (GSSG) inhibits epithelial sodium channel activity in primary alveolar epithelial cells

Charles A Downs et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

Amiloride-sensitive epithelial Na(+) channels (ENaC) regulate fluid balance in the alveoli and are regulated by oxidative stress. Since glutathione (GSH) is the predominant antioxidant in the lungs, we proposed that changes in glutathione redox potential (Eh) would alter cell signaling and have an effect on ENaC open probability (Po). In the present study, we used single channel patch-clamp recordings to examine the effect of oxidative stress, via direct application of glutathione disulfide (GSSG), on ENaC activity. We found a linear decrease in ENaC activity as the GSH/GSSG Eh became less negative (n = 21; P < 0.05). Treatment of 400 μM GSSG to the cell bath significantly decreased ENaC Po from 0.39 ± 0.06 to 0.13 ± 0.05 (n = 8; P < 0.05). Likewise, back-filling recording electrodes with 400 μM GSSG reduced ENaC Po from 0.32 ± 0.08 to 0.17 ± 0.05 (n = 10; P < 0.05), thus implicating GSSG as an important regulatory factor. Biochemical assays indicated that oxidizing potentials promote S-glutathionylation of ENaC and irreversible oxidation of cysteine residues with N-ethylmaleimide blocked the effects of GSSG on ENaC Po. Additionally, real-time imaging studies showed that GSSG impairs alveolar fluid clearance in vivo as opposed to GSH, which did not impair clearance. Taken together, these data show that glutathione Eh is an important determinant of alveolar fluid clearance in vivo.

Keywords: Cys thiol; alveolar flooding; oxidative stress; redox potential.

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Figures

Fig. 1.
Fig. 1.
Extracellular shifts in glutathione (GSH)/glutathione disufide (GSSG) glutathione redox potential (Eh) potential affect epithelial Na+ channels (ENaC) open probability (Po). A: representative portions of a trace taken from a continual cell attached patch-clamp recording of a T2 cell at a holding potential of 10 mV (−Vp) is shown. Closed states (c) are indicated by dashed line and # indicates a break from continual recording. B: point amplitude histograms were constructed from the data for each condition in A. Peaks in the distribution represent 0 or 1 open channel. C: current/voltage relationship of representative trace shown in A, with a calculated conductance (γ = 26.6 pS) that is typical of nonselective cation channels. D: dot plot graph of 12 separate T2 cells shows effects of control (−210 mV; CTR), GSSG (−75 mV), and then GSH (−213 mV) sequentially added on ENaC open probability in continual trace recordings. Individual cells are represented by open circles, and closed circles represent average ± SE. *P < 0.05. E: GSSG (−75 mV) similarly decreases ENaC Po in T1 cells. F: cell attached recordings were performed before (−205-mV redox potential) and after adding a mixture of GSH and GSSG (redox potentials ranging from −300 to −100 mV) to alveolar epithelial cells. Percent control values for individual cells (circles) were calculated by dividing open probability after the addition of GSH/GSSG by the open probability under control conditions and multiplying by 100. Open squares with error bars represent means ± SE for each redox potential. Linear regression was performed to determine the best fit relationship between redox potential and ENaC activity.
Fig. 2.
Fig. 2.
N-ethylmaleimide (NEM) attenuates GSSG inhibition of ENaC. A: representative T2 single channel patch-clamp recording shows no change in ENaC activity during the 10-min observation time period in which cells were pretreated with 1 μM NEM and then 400 μM GSSG (applied where indicated). B: enlarged portion of single channel recordings at 5 min and 16 min post 400 μM GSSG treatment. In A and B, closed states are indicated by “c”, and downward deflections from the closed state indicate channel openings. C: a dot plot graph showing that the Po values did not change significantly from 0.41 ± 0.08 to 0.54 ± 0.12 in 13 independent observations.
Fig. 3.
Fig. 3.
Positive shifts in GSH/GSSG Eh decrease the rate of lung fluid clearance in vivo. C57Bl/6J mice were given a tracheal instillation (5 μl saline/mg body wt) containing either 400 μM GSH (n = 7) or 400 μM GSSG (n = 6). Fluorometric data quantified at 5-min intervals for up to 4 h, with 30-min averaged values reported. Plotted values show the effects of GSH (black circles) and GSSG (black triangles) on relative lung fluid clearance. Symbols represent means ± SE for each condition. Average data were then fit to an exponential model for fluid clearance (see text).
Fig. 4.
Fig. 4.
Changes in extracellular GSH/GSSG Eh alter cellular oxidant production and intracellular redox. A: line graph demonstrating a change in extracellular reactive oxygen species (ROS) from isolated rat T2 cells challenged with a range of redox potentials using Amplex Red for determining H2O2 production. B: images of live lung tissue labeled with dihydroethidium (DHE; as an indicator of O2·− production) and subjected to a highly oxidized redox potential (−75 mV) or control (−205 mV) conditions. C: bar graph quantifying an increase in intracellular ROS production (i.e., DHE intensities) in lung tissue subjected to a highly oxidized redox potential. *P < 0.05.
Fig. 5.
Fig. 5.
α-ENaC is S-glutathionylated. A, left: control Western blot showing α-ENaC detection in R3/1 (full length) and T2 (cleaved) cells. IP, immunoprecipitated; IB, immunoblot. Middle: Western blot analysis of α-ENaC immunoprecipitated samples blotted with anti-GSH antibody indicates direct S-glutathionylation of α-ENaC subunit. Right: IgG signal control. B: control BioGEE assays conducted on equal number T2 cells under control, H2O2, or diamide treatments. Total cell lysate was run on a polyacrylamide gel under reducing (+DTT) and nonreducing (−DTT) conditions and labeled for biotin. Results are representative of 3 independent observations. C: T2 cells were incubated with H2O2 or diamide in the presence of BioGEE then lysed, ran on a gel, and immunoblotted for α-ENaC under reducing and nonreducing conditions. D: quantification of cleaved α-ENaC protein-SSG averaged from 3 independent observations.
Fig. 6.
Fig. 6.
Predicted ENaC subunit S-glutathionylation sites (shown in combination). α: 89C; 159C; 499C; 503C; 507C; 694C. β: 43C; 61C; 89C; 194C; 210C; 247C; 359C; 438C; 534C. γ: 100C; 551C.
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