Acute EtOH consumption increases ROS production in C57/Bl6 mice.

<p>Top panels (A–C) show excised lung from control animals, or mice ingesting 20% EtOH solution for 3 days, following instillation of 200 µL DHE solution. A = 510/620 nm excitation emission (red signal); 30 sec exposure. B = panel A co-registered with x-ray image C = Additive primary color (RGB) image of lungs excised en bloc (showing trachea, heart, and lung. Panels D (510/620 nm; 10 min exposure) and E (510/620 nm co-registered with X-ray) show excised lung from control and EtOH mice following instillation of 200 µL vehicle control. Intensity bars, and rhodamine positive signal controls included for B–E. F) Quantification of homogenized lung samples (shown in A–E) using a microplate reader assay (n = 12 from 3 animals; p<0.05).</p

Multichannel confocal imaging shows EtOH mediated changes in Rac1 expression and subcellular localization in the lung.

<p>Anti-Rac1 antibody detected using Alexa 488 conjugated secondary antibody (488/519 nm; green fluorescence); nuclei were stained DAPI (350/470 nm; blue fluorescence); and plasma membrane labeled with Cell Mask Deep Red (649/666 nm). <u>Right panel</u>: white light image of lung slice preparation and Rac1 labeling; <u>middle panel</u>: Rac1 localization relative to DAPI stained nuclei; <u>left panel</u>: Rac1 co-localization with Deep Red labeled plasma membrane. Pixels containing both red and green color contributions produce various shades of orange and yellow indicate Rac1 co-localization with the plasma membrane. Subsets represent z-axis obtained from horizontal and vertical regions as indicated.</p

Acute EtOH treatment increases ROS production.

<p>A) Lung tissue preparation was obtained from 3 month old Sprague Dawley rats and treated acutely (for 1 hour) with various concentrations of EtOH. Amplex Red labeling indicates that 0.16% EtOH significantly increases H₂O₂ production. n = 9 observations from 3 animals. DHE labeling (B) and quantification (C) confirms that 0.16% EtOH significantly increases H₂O₂ production in lung slice preparations. n = 6 observations from 3 animals.</p

Acute 0.16% ETOH treatment increases sodium channel activity.

<p>A) Continuous single channel recording obtained from primary isolated rat T2 cell treated with 0.16% EtOH after 5 min control recording period as indicated with enlarged portions of the trace. Arrow indicates closed state of channels, with downward deflections from arrow indicating Na movement into the cell. B) Point amplitude histograms show frequency of NSC and HSC activity in representative recording before and after EtOH treatment. C) Conductances (γ) of representative HSC (4.9pS) and NSC (11pS) channels shown in representative trace. D) Number and open probability (NPo) of ENaC reported before and after 0.16% EtOH treatment in 7 cell-attached recording; p<0.05. E-F) I/V curve of all cell recordings with average HSC γ = 6.1 and NSC γ = 22.2.</p

Chronic ethanol ingestion modifies Cys thiols on α-ENaC.

<p>A) <u>Left panel</u> F5M labeling of α-ENaC protein immuno-precipitated from chronic ethanol fed mice (left lane) or maltodextrin control animals (right lane). <u>Middle panel</u> shows bioluminescent detection of α-ENaC using the same membrane shown in left panel. <u>Right panel</u> Co-registration of F5M and ENaC signal (ENaC signal is pseudo-colored and F5M signal contrast reduced in order to enhance co-registration of data obtained from the same blot). B) Normalization of F5M RLU to ENaC expression levels; n = 3; p<0.05.</p

Changes in albumin protein concentration confirm fluorometric data obtained from live animals.

<p>A) C57Bl6 mice challenged by IT instillation of saline, 1 mM amiloride, 1 mg/mL LPS, or 1 mM glibenclamide as indicated. n = 2 for each observation with * = p<0.05. B) C57Bl6 mice (black bars), maltodextrin-control fed mice (white bars); or chronic ethanol mice (grey bars) were treated with 1 mg/mL LPS in the presence or absence of 1 µM NSC23766. n = 3 independent observations with * = p<0.05. In 10A–B, the percent changes in albumin protein concentration were obtained 90 min following instillation; percentages greater than average control values indicate enhanced rates of fluid clearance, whereas negative percent changes are indicative of alveolar flooding.</p

Chronic ethanol ingestion alters protein expression in the lung.

<p>A) Normalized α-ENaC mRNA levels are elevated in C57Bl/6 mouse lung chronically ingesting 20% v/v EtOH. Data represents 9 independent observations from 3 animals in EtOH and isocaloric control groups. B) Expression of α-ENaC protein is significantly increased in mice chronically ingesting 20% w/v EtOH. Ave of data reported from n = 3 mice from EtOH and isocaloric control groups. Data normalized to β-actin expression levels, and p<0.0001. C) Representative western blot of C57Bl/6 mouse lung following chronic alcohol, or isocaloric control diets w/without 1 mg/mL LPS inoculation. D,E) Densimetric analysis show NOX2 catalytic domain gp91^phox (n = 3) and Rac1 (n = 6) significantly increases following (chronic) EtOH and (acute) LPS treatment. p<0.05 where indicated by asterisks.</p

H₂O₂ modifies Cys thiols on α-ENaC.

<p>A) Right panel: α-ENaC protein was heterologously expressed (with β- and γ- subunits) and immunoprecipitated from HEK cells. Left panel: Fluoroscein-5-Maleimide (F5M) labeling of free Cys thiols decreases with oxidizing conditions; excitation emission 480/535 nm; non-reducing gel (−DTT). B) Simple linear regression graph of F5M relative light units (RLU) and H₂O₂ treatment shows relationship between Cys modification and H₂O₂ concentration. Dependent variable: F5M RLU. Independent variable: mM H₂O₂. n = 15; p = 0.05. C) Specificity of F5M label. Right lane = mouse lung homogenate labeled with 0.6 mM F5M. Left lane = mouse lung homogenate labeled with 0.6 mM F5M with/without 6 mM glutathionne. D) F5M RLU normalized to β-actin expression.</p

LPS inoculation and chronic EtOH ingestion enhances the rate of alveolar fluid clearance.

<p>A) In vivo assessment of lung fluid volumes after saline challenge in EtOH (n = 13), isocaloric control (maltodextrin, n = 13) and C57Bl/6 mice (n = 10) indicate similar patterns of lung fluid clearance; albeit an observed delay in clearance in EtOH mice compared to maltodextrin between 3.5–4 hrs following saline challenge. B) In vivo assessment of 1 mg/mL LPS inoculated animals chronically fed an EtOH diet (n = 4) cleared fluid at faster rates compared to isocaloric control groups of animals fed maltodextrin (n = 5) between 190–220 min following saline challenge (as indicated by solid line, p<0.05). LPS inoculation of EtOH animals showed significantly elevated rates of alveolar fluid clearance compared to LPS inoculated C57Bl/6 mice beginning at 20 minutes following saline challenge (denoted by dashed line, p<0.05). C) Co-instillation of 1 µM NSC23766 and LPS in chronic ethanol mice indicates that small G protein Rac1 plays an important role in alcohol lung and alveolar fluid balance. Animals instilled with LPS and NSC23766 (n = 5) failed to clear saline challenge to the extent observed in LPS (n = 4) inoculated EtOH animals; p<0.05 as indicated.</p