Ethyl alcohol (C2H5OH), 190 proof, ACS grade was purchased from Spectrum Chemical (New Brunswick, NJ) and used in all studies below. General chemicals were supplied by Sigma Aldrich (St. Louis, MO) or as indicted.
Ethanol (EtOH) Feeding of Mice
All animal studies were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Emory University Institutional Animal Care and Use Committee (Permit Number: DAR-2001099 & DAR-2001310). All surgery was performed under anesthesia with xylazine and ketamine, and all efforts were made to minimize suffering. A chronic alcohol model was studied using young adult female C57Bl/6 mice purchased from Jackson Laboratory (Bar Harbor, Maine). To start, 6 week old mice were fed 5% v/v EtOH for the first week, with subsequent 5% incremental increases each week until animals reached 20% v/v EtOH. Animals remained on a 20% v/v EtOH diet for an additional 4 weeks in order to model chronic alcohol consumption. Using this general approach, the blood alcohol levels of mice following chronic ethanol consumption has been reported to be 400 mg/dL 
. An age-matched, control group of animals were fed an isocaloric maltodextrin diet. Both EtOH and control animals were given standard chow ad libitum
; dietary intake was comparable in both groups. The benefits of using a chronic mouse model for alcohol abuse has been recently reviewed in 
Acute Ethanol Treatment
Freshly isolated rat alveolar epithelial cells were treated with 0, 0.04%, 0.08%, or 0.16% v/v EtOH in a vehicle solution for 1 hour. The protocol for isolated alveolar cells has been optimized using this rodent model (see below). C57Bl6 mice ingesting 20% v/v EtOH for 3 days modeled the effects of acute alcohol consumption (i.e. binge drinking) in vivo and complements findings using chronic C57Bl6 mice described above.
Primary Cell Isolation
Primary alveolar epithelial cells were isolated from male Sprague Dawley rats purchased from Charles River (Wilmington, MA) as described in 
Western Blot Analysis
Whole lung homogenate was prepared in order to quantify proteins of interest from chronic alcohol mice. Briefly, mouse lung was perfused via the pulmonary artery with phosphate buffer saline (PBS). The lungs were removed en bloc
, rinsed with PBS, and placed on ice prior to homogenization in 3 mL RIPA buffer in mM: 150 NaCl, 10 Na3
, 0.15 SDS, 1% Nonidet P-40, and 0.25% Na deoxycholate. Approximately 100 µg lung homogenate was electrophoresed on a 10% polyacrylamide gel in the presence or absence of DTT, as indicated. Protein was then transferred to Protran nitrocellulose membrane (Schleicher and Schuell BioScience) for immunodetection of protein of interest. Nitrocellulose was blocked in TBST buffer (10 mM Tris, pH 7.5, 70 mM NaCl, and 0.1% Tween) with 5% dry milk prior to incubation with primary antibodies of interest, as indicated. The α-ENaC-C20 antibody was purchased from Santa Cruz; antibodies for NOX2 catalytic and regulatory subunits were purchased from Millipore Upstate; and anti-Rac1 antibody was obtained from Santa Cruz. All membranes were incubated with respective antibody for 1 hr at RT, and washed extensively in TBST buffer prior to incubation with IgG alkaline phosphatase (AP) labeled secondary antibody (KPL, Inc.) at a 1
10,000 fold dilution in TBST for 1 hr at RT. AP luminescent signal was detected using Nitro-Block chemiluminescence enhancer and CDP-Star substrate (Tropix) and Carestream Image Station Gel Logic 4000 Pro (Carestream Health) and compatible imaging software.
Real Time PCR
Total RNA was extracted from whole lung using the RNeasy isolation kit (Qiagen) per manufacturer protocol. RNA was treated with DNase 1 and reverse transcribed using Superscript II RNaseH-reverse transcriptase (Invitrogen). The following pairs of primers were used: α-ENaC forward TGCTCCTGTCACTTCAGCAC, reverse CCCCTTGCTTAGCCTGTT C; β-ENaC forward CCCCTGATCGCATAATCCTA, reverse GCCCCAGTTGAAGATGTAGC; γ-ENaC forward ACCCTTTCATCGAAGACGTG, reverse CCTCTGTGCACTGGCTGTAA; p22phox forward AACGAGCAGGCGCTGGCGTCCG, reverse GCTTGGGCTCGATGGGCGTCCACT; gp91phox forward CAGGAACCTCACTTTCCATAAGAT, reverse AACGTTGAAGAGATGTGCAATTGT; p67phox forward TCCAACGAGGGATGCTCTAC, reverse CACAGGCAAACAGCTTGAAC; gapdh forward CAAGGTCATCCATGACAACTTTG, reverse GGCCATCCACAGTCTTCTGG. Primer pairs were purchased from Integrated DNA Technologies (Coralville, IA). Threshold levels of mRNA expression (ΔΔCt) were normalized to mouse GAPDH levels, and values represent the mean of triplicate samples ± SEM. Data are representative of 3 independent studies.
Single Channel Patch Clamp Analysis and Conductances
Cell-attached single channel patch clamp analysis was performed as recently described in 
. Current-voltage relationships were plotted and chord conductances obtained by calculating the slope of points between hyperpolarizing potentials for HSC channels and between hyperpolarizing and depolarizing potentials for NSC channels. R-squared values are as indicated.
Densimetric Evaluation of Lung Fluid Volume
We determined lung fluid volumes using an in vivo
radiographic imaging assay recently described in 
. Briefly, animals were given a tracheal instillation of saline (5 µL/g body weight) and then placed onto the platform of an in vivo
Multispectral Imaging Station (Carestream Health) in order to determine the rate of fluid clearance in freely breathing, anesthetized mice. Animals were immobilized in the imaging station with continual delivery of isoflurane (1.5 L/min) mixed with 100% oxygen during the imaging studies. Animals were X-rayed at 5 minute intervals up to 240 min with an acquisition period of 120 sec. X-ray settings were set at 2×2 binning, 180 mm field of view, 149 µA X-ray current, 35 k Vp, and 0.4 mm aluminum filter. X-ray density was analyzed using Carestream Health MI software as described in 
Fluorescent Reactive Oxygen Species (ROS) Detection
Three separate yet complementary approaches were used to measure ROS levels following EtOH treatment. Thin 175 micron lung slices were prepared as described in 
prior to incubation in 10 µM dihydroethidium (DHE; Invitrogen) at 37°C for 30 min protected from light. We and others 
, have recently reported that fluorescent detection of the reaction product between O2−
and DHE (generating 2-hydroxyethidium) is a reliable measure of superoxide production in cells. Fluorescence detection of DHE oxidation was analyzed by confocal microscopy (excitation/emission 520/610 nm) as previously reported in 
. DHE fluorescence intensities of lung slices were quantified using Image J, a public domain, Java-based image processing program developed at the National Institutes of Health.
200 µL DHE, or vehicle control, was tracheally instilled in C57Bl/6 lung of animals on control or acute EtOH diet, as described above. Approximately 30 min following DHE instillation, lungs were removed en bloc, and fluorescent detection of ROS was assayed and quantified using Carestream Health In Vivo Multispectral Imaging Station or standard plate reader with 520/625 nm excitation/emission.
Because superoxide is quickly dismutated to H2O2, we used Amplex Red Enzyme Assays (Invitrogen) as an additional measure of ROS release. Lung slices were acutely treated with EtOH. Afterwards, ROS released into the extracellular solution was incubated with Amplex Red reagent solution, prepared per manufacturer’s protocol, for 30 min at 37°C protected from light. Fluorescence intensity of Amplex Red oxidation of H2O2 was determined using a microplate reader (excitation/emission 530/590 nm).
Immunohistochemistry and Confocal Imaging
Unfixed tissue slices were incubated in primary rabbit anti-Rac1 antibody (Santa Cruz) diluted 1
1000, followed by incubation in secondary anti-rabbit IgG conjugated to Alexa 488 (Invitrogen) diluted 1
50,000 in PBS containing 1% BSA and 1× sodium azide. Cell Mask Deep Red plasma membrane stain (Invitrogen) was used to mark subcellular localization of Rac1 and was used per manufacturer protocol. Lung tissue was fixed in 4% paraformaldehyde and mounted in VECTASHIELD HardSet mounting medium with 4′6′-diamidino-2-phenylindole (Vector Laboratories). Confocal imaging was conducted at the Emory-Children’s Pediatric Research Cell Imaging Core using an Olympus FV1000 confocal laser scanning microscope.
cDNA Plasmid Transfection in Human Embryonic Kidney (HEK) Cells
Subconfluent HEK cells were cultured in DMEM/F12 50/50 media (Invitrogen) supplemented with 10% FBS, 2 mM L-glutamine, and 20 U/ml each of penicillin and streptomycin, 83.75 µM gentamycin and 1 µM dexamethasone. Cells were transfected with Lipofectamine 2000 (Invitrogen) per manufacturer’s protocol using 1 µg each α-, β-, γ- rENaC DNA plasmid constructs.
Maleimide Labeling of Reduced Cys
Fluoroscein-5-Maleimide (F5M; Invitrogen) was used to confirm the oxidant sensitivity of lung ENaC. Briefly, endogenous α-ENaC subunit was immunoprecipitated (IPed) from male Sprague Dawley rats, chronic ethanol mice, (isolated as described in 
) or from HEK cells over-expressing HA-tagged α-rENaC construct, alongside β-rENaC, and γ-rENaC subunits for normal channel assembly. Native protein from rat alveolar T2 cells was treated with 0, 0.04%, 0.08%, or 0.16% EtOH, whereas heterologously expressed α-ENaC was subjected to oxidation with 0, 0.1, 0.5, 1.0, and 3.0 mM H2
. Both EtOH and H2
treatment were for 15 min at 37°C in a humidified 5% CO2
incubator. Then, 50 µL of 0.6 mM F5M (reconstituted in 20 mM Tris-HCl (pH 7.4), 0.1 mM MgCl2
, and 1 mM MnCl2
) was applied to 50 µL protein in standard RIPA buffer. For signal control studies, 0.6 mM F5M was pre-incubated with 6 mM glutathione. In all experiments, F5M reaction was quenched by adding equal volume 5X sample buffer containing 100 mM DTT. Samples were then boiled (95°C) for 10 min, spun down, and then electrophoresed on a 10% acrylamide gel. F5M labeled protein was transferred onto nitrocellulose and detected using an FX-Pro Multispectral Imager (Carestream Health) with 480/535 nm excitation/emission filters. Immuno-detection of α-ENaC was completed as described above. Fluorescent F5M and luminescent α-ENaC signals were contrasted, pseudo-colored, and over-laid using Carestream Health MI software.
Evan’s Blue-labeled Albumin Measurements
C57Bl/6, maltodextrin-fed, and chronic ethanol mice were tracheally instilled with solution containing 5% bovine serum albumin (BSA) and Evans blue dye (0.1 mg/mL). Alveolar fluid clearance (AFC) was assessed 90 min after instillation by measuring the change in concentrations of alveolar Evans blue-labeled albumin. AFC was calculated as follows, and as described in 
[(Vi-Vf)/Vi] X 100, where Vi is the volume of instilled Evans blue-labeled albumin and Vf is the final alveolar fluid calculated as follows: Vf
(Vi X EBi)/EBf, where EB is the concentration of Evans blue-labeled albumin in the instilled solution (i) and the final alveolar fluid (f). Protein concentration was determined with the use of a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE).
Data summarized as means ±SE. Single comparisons were performed using Student’s t
-test, whereas multiple comparisons were performed using one-way ANOVA followed by Holms test for pairwise comparisons. Linear and non-linear regression analysis was performed and plotted using Sigma Plot 10 software. Post-hoc power analysis was performed using Statistical Analysis System (SAS 9.3) software, which indicated that in vivo
sample sizes are adequate for an effect with >80% power using traditional significance criterion (alpha