Am J Physiol Lung Cell Mol Physiol 298: L509–L520, 2010.
First published January 22, 2010; doi:10.1152/ajplung.00230.2009.
Rac1-mediated NADPH oxidase release of O⫺
2 regulates epithelial sodium
channel activity in the alveolar epithelium
Yoshizumi Takemura,2 Preston Goodson,2 Hui Fang Bao,2 Lucky Jain,1 and My N. Helms2
Departments of 1Pediatrics and 2Physiology, Emory University, Atlanta, Georgia
Submitted 13 July 2009; accepted in final form 18 January 2010
redox; NOX2; lung slice; fluorescence-activated cell sorting; singlechannel patch
such as superoxide anions (O⫺
2 ), are
important secondary messengers involved in maintaining normal cell function. Although release of O⫺
2 typically occurs in
the mitochondria (as an incidental by-product of metabolic
respiration), the identification of novel NADPH oxidase
(NOX) isoforms in nonphagocytic cells has piqued new interest in the study of O⫺
2 signaling. In general, NOX is responsible
for transporting electrons across biological membranes to reduce molecular oxygen to O⫺
2 (reviewed in Ref. 1). Of the
seven homologs of NOX characterized thus far (18), NOX2, also
known as gp91phox, is the best studied member of the NOX
family. In Fig. 1, we diagram the general mechanism of NOX2
activation by the small G protein, Rac1, as it is understood
to occur in phagocytic and nonphagocytic cells. Essentially,
NOX2 production of O⫺
2 requires Rac1 activation of cytoplasREACTIVE OXYGEN SPECIES,
Address for reprint requests and other correspondence: M. N. Helms, 615
Michael St., Whitehead Biomedical Research Bldg., Suite 646, Atlanta, GA
30322 (e-mail: [email protected]).
http://www.ajplung.org
mic subunits (p47phox, p67phox, and p40phox). The translocation
and assembly of these regulatory subunits to the catalytic
domain, also termed NOX2, stimulates electron transfer from
NADPH to oxygen. Following electron transfer, O⫺
2 are released into the extracellular space. In the schematic, we depict
NOX2 in the apical membrane, near epithelial sodium channels
(ENaCs), to reflect our hypothesis that Rac1-mediated NOX2
release of O⫺
2 regulates ENaC activity. Moreover, we explore
the signal transduction cascade of Rac1 in lung cells, as it
relates to ion channel function. Although we (12) have previously reported that O⫺
2 plays an important role in regulating
lung ENaC, the signaling mechanisms responsible for altered
sodium transport remain unclear. Additionally, the physiological role of nonphagocytic NOX2 in the lung has not been
established.
In the current study, we examine the role of NOX in the
signal transduction cascade leading to normal sodium channel
activity in the alveolar epithelium, which has not been previously described. ENaCs play a key role in maintaining alveolar
fluid balance in the lung by creating the osmotic driving force
needed to move water out of the air space. Although the precise
relationship between O⫺
2 release and ENaC function has not
been clearly defined, it is certain that both NOX and ENaC
activity are reliant on small monomeric G protein signaling.
Our studies described below provide a plausible link between
small G protein signaling, O⫺
2 production, and ENaC regulation in alveolar cells. The interrelatedness of these signaling
proteins presents a novel mechanism of lung ENaC regulation.
The alveolar epithelium is made up of alveolar type 1 and
type 2 cells. Both cell types express functional ENaCs; however, our recent studies indicate that type 1 and 2 cells may
differ significantly in response to oxidative stress (12). Additionally, fundamental comparisons between type 1 and 2 cells,
such as differences in the level of NOX expression, have not
been made in these neighboring cells. Moreover, a functional
role for alveolar NOX has not been clearly described. There
are, however, several correlations between changes in oxidative state and the ability of the lung to maintain salt and water
homeostasis, indicating that regulated NOX output of O⫺
2 must
be important in ENaC regulation. For instance, high-altitude
pulmonary edema (HAPE) is a serious condition affecting the
ability of the lung to exchange CO2 for oxygen due to excessive fluid accumulation in low PO2 environments. The low and
limited availability of molecular oxygen slows the rate of O⫺
2
production and, presumably, alters the normal function of
ENaC and other important processes related to maintaining
homeostasis. Additionally, patients with chronic granulomatous disease (CGD) inherit mutations in the normal expression
of NOX enzyme, are immunocompromised, and also suffer
from severe pneumonia. Clearly, in CGD, the compromised
ability to release O⫺
2 impacts the ability of the lung to clear
1040-0605/10 $8.00 Copyright © 2010 the American Physiological Society
L509
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Takemura Y, Goodson P, Bao HF, Jain L, Helms MN. Rac1mediated NADPH oxidase release of O⫺
2 regulates epithelial sodium
channel activity in the alveolar epithelium. Am J Physiol Lung Cell
Mol Physiol 298: L509 –L520, 2010. First published January 22,
2010; doi:10.1152/ajplung.00230.2009.—We examine whether alveolar cells can control release of O⫺
2 through regulated NADPH
oxidase (NOX) 2 (NOX2) activity to maintain lung fluid homeostasis.
Using FACS to purify alveolar epithelial cells, we show that type 1
cells robustly express each of the critical NOX components that
phox
catalyze the production of O⫺
, p22phox, p67phox,
2 (NOX2 or gp91
phox
phox
p47
, and p40
subunits) as well as Rac1 at substantially higher
levels than type 2 cells. Immunohistochemical labeling of lung tissue
shows that Rac1 expression is cytoplasmic and resides near the apical
surface of type 1 cells, whereas NOX2 coimmunoprecipitates with
epithelial sodium channel (ENaC). Since Rac1 is a known regulator of
NOX2, and hence O⫺
2 release, we tested whether inhibition or activation of Rac1 influenced ENaC activity. Indeed, 1 ␮M NSC23766
inhibition of Rac1 decreased O⫺
2 output in lung cells and significantly
decreased ENaC activity from 0.87 ⫾ 0.16 to 0.52 ⫾ 0.16 [mean
number of channels (N) and single-channel open probability (Po)
(NPo) ⫾ SE, n ⫽ 6; P ⬍ 0.05] in type 2 cells. NSC23766 (10 ␮M)
decreased ENaC NPo from 1.16 ⫾ 0.27 to 0.38 ⫾ 0.10 (n ⫽ 6 in type
1 cells). Conversely, 10 ng/ml EGF (a known stimulator of both Rac1
and O⫺
2 release) increased ENaC NPo values in both type 1 and 2
cells. NPo values increased from 0.48 ⫾ 0.21 to 0.91 ⫾ 0.28 in type
2 cells (P ⬍ 0.05; n ⫽ 10). In type 1 cells, ENaC activity also
significantly increased from 0.40 ⫾ 0.15 to 0.60 ⫾ 0.23 following EGF
treatment (n ⫽ 7). Sequestering O⫺
2 using 2,2,6,6-tetramethylpiperidineN-oxyl (TEMPO) compound prevented EGF activation of ENaC in both
type 1 and 2 cells. In conclusion, we report that Rac1-mediated NOX2
activity is an important component in O⫺
2 regulation of ENaC.
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Rac1-MEDIATED NOX REGULATES LUNG ENaC
study, we examine NOX-mediated O⫺
2 release, following small
G protein activation, as a new and important signaling mechanism that overlaps with canonical ENaC regulatory pathways.
METHODS
fluid. Because sodium channels are the rate-limiting factor in
net fluid reuptake, there must be an important relationship
between O⫺
2 signaling and the ENaC regulatory pathways,
particularly in the lungs.
ENaCs are located in the apical membrane of polarized cells
and serve primarily to transport Na from the lumen to the
interstitial space and ultimately back into the bloodstream. The
net movement of Na in this direction creates the osmotic
driving force needed for lung fluid clearance. The importance
of ENaC in maintaining homeostasis and viability is best
appreciated in ENaC knockout mice: low expression of
␤-ENaC significantly impairs lung fluid clearance (25), and
␣-ENaC knockout animals die within 40 h of birth due to an
inability to clear lung fluid (14). Although it is clear that
normal ENaC function is critical, the precise mechanism of
sodium channel regulation remains unknown.
Recently, we (28) reported that steroid hormones, such as
aldosterone and dexamethasone, regulate O⫺
2 production. This
established a rudimentary connection between ENaC and O⫺
2
signaling, given that corticosteroids are the principal hormonal
regulators of ENaC function. We provided a stronger link,
using 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) compound, which scavenged cellular O⫺
2 and led to notable decreases in ENaC activity (29). Conversely, increasing local O⫺
2
levels in both A6 and primary lung cell models resulted in
significant increases in single-channel measurements (12, 29).
Together, these studies indicate that regulated O⫺
2 release cross
talks with ENaC signal transduction pathways. In the present
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Fig. 1. Schematic of NADPH oxidase (NOX) complex signaling and epithelial
sodium channel (ENaC) in apical membrane. NOX2 is a multiprotein enzyme
complex with the catalytic subunit (NOX2) stabilized at the cellular membrane
via association with p22phox subunit. Complete activation of the catalytic
domain requires appropriate assembly of cytoplasmic p47phox, p67phox, and
p40phox subunits. These cytoplasmic subunits are under the control of monomeric G protein, Rac1, which cycles between an active GTP-bound state and
inactive GDP-bound state [regulated by guanine nucleotide exchange factors
(GEFs)], which exchanges GTP for GDP. This figure depicts activated NOX2
complex proximal to ENaCs. P, phosphorylated; 2e⫺, 2 electrons.
Animal care. Male Sprague-Dawley rats were housed with access
to standard rat diet and water ad libitum. Between weeks 8 and 12,
animals were anesthetized and killed for experimentation in accordance with Institutional Animal Care and Use Committee (IACUC)
guidelines. All animal protocols conform to National Institutes of
Health animal care and use guidelines and were approved by Emory
University IACUC.
FACS of type 1 and 2 cells. Rat lungs were lavaged with 2 mg/ml
DNase and 1.5 mg/ml elastase dissolved in RPMI/HEPES solution.
The lungs were then minced into small 1.5- to 2-mm pieces before
filtering through a 40-␮m filter. Cells were incubated with 1 mg/ml rat
IgG antibody and goat anti-rat IgG magnetic beads before elution
through an LC column (Miltenyi Biotec) to remove macrophages
(leaving mostly type 1 and 2 cells in the eluted buffer). Eluted cells
were centrifuged and resuspended in flow sort buffer (2% FBS in Caand Mg-free PBS) at a concentration of 1 ⫻ 106 cells/ml. Single cell
suspension of type 1 and 2 cells were labeled using a 1:1,000 dilution
of LysoTracker Red (Invitrogen) and Fluorescein-labeled Erythrina
crista-galli Lectin (ECL; Vector Laboratories). In the flow cytometer
(FACSVantage SE; Becton Dickinson), ECL-bound type 1 cells are
extrapolated based on their unique forward (FSC) and fluorescent
(FL1) scatter profiles following argon laser 488-nm excitation. From
the same sort sample, type 2 cells with distinct FSC and FL2
scattering (on 633-nm laser excitation) were simultaneously sorted
from the mix population of cells.
Western blot analysis. Flow-sorted pneumocytes were rinsed three
times with ice-cold PBS supplemented with 1⫻ protease inhibitors.
Cells were pelleted and then lysed in 600-␮l RIPA buffer (150 mM
NaCl, 10 mM NaPO4, pH 7.4, 0.1% SDS, 1% Nonidet P-40, 0.25%
Na⫹-deoxycholate). All protein were electrophoresed on 7.5 or 15%
acrylamide gels (where appropriate) under denaturing condition. Protein lysates were then transferred to Protran nitrocellulose membrane
(Schleicher & Schuell) for immunolabeling. The membrane was
blocked in TBST buffer (10 mM Tris, pH 7.5, 70 mM NaCl, and 0.1%
Tween) with 5% dry milk and then incubated with 1 ␮g/ml NOX2
(Upstate Biotechnology), p67phox (Millipore), p47phox (Millipore),
p40phox (Millipore), p22phox (Santa Cruz Biotechnology, Santa Cruz,
CA), or Rac1 (Santa Cruz Biotechnology) antibodies for 1 h at room
temperature. IgG-alkaline phosphatase (AP)-labeled secondary antibody (KPL, Gaithersburg, MD) was added at a concentration of 1
␮g/10 ml TBST and incubated for another 1 h at room temperature.
After five TBST washes, AP signal was detected using Nitro-Block
chemiluminescence enhancer and CDP-Star substrate (Tropix) in
combination with Kodak Image Station 2000MM and Carestream MI
software.
Immunoprecipitation. A heterogeneous mixture of primary rat type
1 and 2 cells was used in coimmunoprecipitation (co-IP) studies.
Briefly, 20 ␮g of rabbit anti-NOX antibody (Upstate Biotechnology)
or goat anti-␣-ENaC COOH-terminal antibody (Santa Cruz Biotechnology) was incubated in 3-mg lung-cell RIPA buffer-lysate overnight. Antibody-bound protein complexes were pulled down using
300 ␮l of IgG resin (UltraLink Immobilized Protein A Plus beads;
Pierce) in 500-␮l lysate volume. Following IP, standard Western blot
procedures (described above) were used to detect NOX2 co-IP with
␣-ENaC subunit.
Lung slice preparation. Lung slices that were 250-␮m thin were
prepared from rat lungs as previously described (13). Briefly, after
pulmonary perfusion, low-melting-point agarose was instilled via the
trachea. Excised lungs were removed en bloc, iced, and then mounted
onto a vibrating microtome to prepare tissue slices. Tissue slices were
transferred to 50:50 ice-cold DMEM/F-12 media (containing 10%
Rac1-MEDIATED NOX REGULATES LUNG ENaC
CaCl2 · 2H2O, and 1.2 MgSO4 · 7H2O) following NSC23766 or vehicle
treatment. DHE (10 ␮M) incubated with cells at 37°C for 20 min
shielded from light. The cells were harvested for HPLC analysis
(using the Beckman HPLC System Gold) in 300 ␮l of methanol,
homogenized, and then passed through a 0.22-␮m filter. As an
alternative to HPLC detection of O⫺
2 release, the fluorescence property of oxyethidium was additionally measured using a Synergy 4
(Biotek) microplate reader with the appropriate 520-/610-nm filter
sets.
Statistical evaluation. Statistical determinations were made by
paired t-test analysis with P ⬍ 0.05 considered significant. Standard
errors of the means (SE) were reported.
RESULTS
FACS of type 1 and 2 cells. Detailed study of type 1 cell
property and function has been limited, largely due to the
technical difficulties associated with isolating a pure preparation of primary type 1 cells (described further in Refs. 3, 16).
Furthermore, an ideal cell line for studying type 1 cells has not
been firmly established. As such, a “type 1-like” model system,
in which primary isolated type 2 cells remain in culture past
day 7 (and presumably transform into a type 1 phenotype), is
commonly employed in biochemical assays (4). It is not clear
how representative this type 1-like model system can be of
alveolar cells in vivo. In the current study, we isolate primary
type 1 and 2 cells simultaneously, and with certain purity,
using standard flow cytometric techniques. Essentially, type 1
and 2 cells were FACS-sorted based on their differential
binding to vital dyes (previously characterized in Ref. 12) and
innate differences in size (10). Because type 1 and 2 cells
differentially bind vital markers fluorescein-labeled ECL and
LysoTracker Red, respectively, with specificity, and because
type 1 cells are significantly larger than cuboidal type 2 cells,
FACS sorting is a quick and reliable approach to isolating each
cell type.
Figure 2A is a grayscale image of a surfactant labeled type
2 cell (inset) and a type 1 cell labeled with RTI40 antibody.
Fig. 2. FACS of alveolar type 1 and 2 cells. A: flow cytometry sorts heterogeneous lung cells based on size and fluorescent marker binding. The inset (top left)
shows an type 2 cell, which are small and cuboidal cells with basal surface area averaging 180 ␮m2 (reviewed in Ref. 10). Type 1 cells are significantly larger,
with morphometric data indicating that the membrane surface area averages 5,000 ␮m2 (reviewed in Ref. 10). B: side light scatter (SSC) and forward light scatter
(FSC) dot blots of single suspension lung cells. FSC is indicative of cell size, and SSC is indicative of granularity. Of the large cells gated in G1, we selected
cells that were additionally bound to Erythrina crista-galli Lectin (ECL), which is a vital dye that binds to type 1 cells with selectivity. The cells gated in G2
were additionally sorted for LysoTracker Red (LTR) fluorescence. C: fluorescence 1 [FL1; enhanced green fluorescent protein (eGFP)] vs. fluorescence 2 (FL2; LTR)
dot blot of lung cells. The dot blot shows the distinct scatter profiles of type 1 cells [in region 1 (R1)] and 2 cells [in region 2 (R2)]. Postsort analysis confirmed
similar fluorescent and FSC scatter profiles of isolated type 1 and 2 cells (data not shown). FS, forward scatter; SS, side scatter; H, height.
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FBS, 2 mM L-glutamine, 1 ␮M dexamethasone, 84 ␮M gentamicin,
and 20 U/ml penicillin-streptomycin) and used within 6 h for patchclamp analysis.
Confocal imaging. Lung tissue, fixed in 2% paraformaldehyde
solution, was immunolabeled with Rac1 or NOX2 antibodies (listed
above). Primary antibodies were diluted 1:100, whereas secondary
antibodies (as indicated in figure legends) were diluted 1:50,000 in
PBS containing 1% BSA and 1⫻ sodium azide (PBS/BSA). All
washes were performed with PBS/BSA before mounting with
VECTASHIELD HardSet mounting medium with 4=,6=-diamidino-2phenylindole (DAPI; Vector Laboratories), although DAPI signals are
not shown. Thin optical sections (1-␮m z-stack images) were obtained
using an Olympus BX61WI microscope designed for confocal fluorescence observations alongside Fluoview FV10-ASW 1.7 software.
Cell-attached patch-clamp. Lung slices were rinsed and then incubated in patch-clamp solution containing 140 mM NaCl, 5 mM KCl,
1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.4. Gigaohm
seals were formed on alveolar epithelial cells using a glass microelectrode back-filled with patch solution. An Axopatch-1D (Molecular
Devices) amplifier interfaced through an analog-to-digital board to a
personal computer collected single-channel data. Channel currents
were recorded at 5 kHz and filtered at 1 kHz with a low-pass Bessel
filter. We used the product of the number of channels (N) and the
single-channel open probability (Po) as a measure of ENaC activity
within a patch. NPo was calculated using Fetchan and Clampfit 10.1
software (Molecular Devices).
Pharmacological regulators of Rac1. EGF and NSC23766, reagents that activate and inhibit Rac1, respectively, were purchased
from Calbiochem. The superoxide dismutase mimetic TEMPO was
purchased from Sigma-Aldrich.
⫺
HPLC analysis and O⫺
2 measurements. The reaction product of O2
with dihydroethidium (DHE) yields a fluorescent product, 2-hydroxyethidium (2-OH-E⫹), measureable both by its fluorescent properties
(excitation 520 nm/emission 610 nm) and elution time in HPLC
assays. HPLC measurements of O⫺
2 release were performed in the
Free Radicals in Medicine Core, Division of Cardiology, Department
of Medicine, Emory University, as previously described (8). Briefly,
A6 model cells were grown on 100-mm plastic dishes (Corning) and
washed with Krebs-HEPES buffer, pH 7.35 (in mM: 99 NaCl, 4.69
KCl, 25 NaHCO3, 1 KH2PO4, 5.6 D-glucose, 20 Na-HEPES, 2.5
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Rac1-MEDIATED NOX REGULATES LUNG ENaC
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Fig. 3. Western blot analysis of type 1 (T1) and 2 (T2) cell purity. A: 200,000
type 1 and 2 cells, obtained using FACS, were lysed and analyzed using
standard polyacrylamide gel electrophoresis. In both A and B, lane 1 ⫽
FACS-isolated type 1 cells, and lane 2 ⫽ FACS sorted type 2 cells. A shows
that only lane 1 is immunoreactive with anti-rat type 1 antibody (RTI40).
Conversely, B shows that only lane 2 is immunoreactive with surfactant
protein C (SP-C) antibody.
relative values are shown in Fig. 4B (quantified from 3 independent cell sorts and Western blot experiments). In each
study, Rac1, NOX2, p67phox, and p47phox expression in type 1
cells are ⱖ8-fold greater than in type 2 cells, whereas p40phox
and p21phox levels are ⱖ20-fold greater in type 1 cells compared with type 2. Below, we investigated the subcellular
localization of Rac1 and NOX2 (catalytic domain) using intact
lung tissue slices.
NOX2 and Rac1 labeling in lung tissue. Immunohistochemical labeling of lung tissue slices show predominant Rac1 and
NOX2 localization in type 1 cells. The left panels in Fig. 5 are
all bright field images of intact alveoli under ⫻40 magnification. In Fig. 5, A and B, type 1 cells were positively identified
using fluorescein-labeled ECL (green cells; right); Rac1 (Fig. 5A)
and NOX2 (Fig. 5B) were colabeled with ECL using antirabbit secondary antibody conjugated to Alexa Fluor 568 (red
signal). In Fig. 5, C and D, alveolar cells were labeled with
LysoTracker Red to highlight type 2 cells, with Rac1 (Fig. 5C)
and NOX2 (Fig. 5D) counter-labeled using rabbit secondary
antibody conjugated to Alexa Fluor 488 (pseudolabeled cyan
for clarity and in no way altering data output). Regardless of
the detection antibodies used, these images show that Rac1 and
NOX2 are more readily detectable in type 1 cells, over type 2
cells, in intact alveoli. Seemingly, NOX2 expression is near the
luminal surface of the alveoli, where, interestingly, ligandgated Na channels are known to reside.
NOX2 co-IP with ENaC. We confirmed that the catalytic
domain of NOX2 and ENaC resides proximal to each other in
the apical membrane using standard IP studies. In Fig. 6, lane 1,
␣-ENaC antibody (generated in goat against the COOH-terminal domain of ␣-subunit) was used to pull down ENaCs. The
second lane served as a signal control, where anti-NOX2
antibody was used to IP the NOX catalytic domain. Standard
Western blot analysis, using rabbit anti-NOX antibody, shows
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This figure illustrates the morphological differences between
the two cell types that make up the alveolar surface area.
Macrophages were removed before cell sorting using standard
immunoadsorption techniques described above in METHODS.
Figure 2B shows the side light scatter (SSC) and FSC dot blots
of single suspension lung cells as they pass through the flow
cytometer. The magnitude of the forward scatter is roughly
proportional to cell size. Thus the smaller type 2 cells are
located in the bottom left corner [gate 2 (G2)], and type 1 cells
are in G1. Side scatter profiles show granularity; cells in the top
left and right quadrants are either dead cells or neutrophils,
respectively, and were not gated for further analysis. Very
small particles were ignored in threshold settings. Figure 2C
shows the two-color dot-plot experiments performed using
compatible fluorescent dyes. We (12) have recently reported
that LysoTracker Red binds to lamellar bodies found in type 2
cells (and not in type 1 cells). Additionally, we (12, 13)
routinely report the use of fluorescein-labeled ECL, which
fluoresces green, as a reliable marker of type 1 cell surface
protein. The major population of cells in region 1 represents
the type 1 cells bound to ECL, with discrete 488-nm (green)
emission. Type 2 cells with bright red fluorescence are in
region 2. Multiparametric analysis of cells, obtainable via
FACS sorting, is necessary for the precise physical separation
of type 1 and 2 cells from a mix lung cell preparation. In the
cytometer, ECL-bound type 1 cells are extrapolated based on
both their far forward profile and fluorescence following argon
laser 488-nm excitation. From the same sort sample, type 2
cells with distinct forward scatter profiles and fluorescent
scatter (on 633-nm laser excitation) are simultaneously sorted.
The FACS sort rejects cells that are double positive for ECL
and LysoTracker Red bound cells, which are visible in the top
right quadrant of Fig. 2C. These multiparametric analyses
make FACS an efficient and reliable method for isolating type
1 and 2 cells for further biochemical studies. Cells were
subjected to the FACS again to verify that only a single
population of cell was indeed collected in postsort analysis. For
example, FL1_GFP vs. FSC histograms were generated for
cells collected from G1, to verify only cells with type 1
characteristics had been collected (data not shown).
Western blot analysis of FACS-isolated cells served as
another important determinant of sample purity. In Fig. 3,
equal cell number of type 1 and 2 cells were loaded onto
polyacrylamide gels for detection of rat type 1 protein (40 kDa)
and surfactant protein C (SP-C; 21 kDa). The RTI40 antibody
was developed by Dobbs et al. as a specific type 1 cell marker,
and SP-C is the most specific label for type 2 cells (15). As
expected, FACS-sorted type 1 cells (Fig. 3A; lane 1) were
immunoreactive with rat type 1 specific antibody, and type 2
cells in lane 2 were not. Flow-sorted type 2 cells, however,
were immunoreactive with anti-SP-C antibody in Fig. 3B; type
1 cells in lane 1 were negative. Because there is no crosscontamination of cell types, we were then able to make novel
biochemical comparisons between type 1 and 2 cells.
Type 1 cells robustly express NOX2. To establish a physiological role for NOX2 release of O⫺
2 in lung ENaC regulation,
we first determined where and how much NOX2 was expressed
in the alveoli. In Fig. 4A, equal number FACS-sorted type 1
and 2 cells were lysed and immunoblotted for each NOX
subunit and Rac1. We found that type 1 cells express NOX2
and Rac1 at substantially higher levels than type 2 cells; the
Rac1-MEDIATED NOX REGULATES LUNG ENaC
L513
that ENaC associates with NOX2 in primary rat alveolar
epithelial cells. Figure 6 is representative of four independent
studies wherein NOX2 pulled down with ENaC. To be certain
the signal generated is not from nonspecific binding of the
conjugated secondary antibody, we subjected IP samples to
Western blot analysis in which the blotted membrane was
incubated with horseradish peroxidase-labeled anti-rabbit secondary antibody only, the result of which was negative (data
not shown).
A new role for Rac1-mediated NOX signaling in maintaining
lung fluid homeostasis. Monomeric G proteins have long been
implicated in regulating ENaC (7), however, the precise mechanism of G protein activation remains unclear. Using cellattached patch-clamp analysis and compounds that alter Rac1
activity, we show acute regulation of ENaC via Rac1-mediated
NOX2 signaling in type 1 and 2 cells in situ.
In all patch-clamp recordings, we sample control recording
periods for 5 min before drug application. As such, the same
patch-clamp recording can be used as its own control. This
sampling time also adequately reflects channel activity (NPo)
without making assumptions about the total N present in a
patch or the Po of a single channel (previously described in
Ref. 22). Figure 7A shows a continuous current trace obtained
from a representative type 1 cell recording that was accessed
from live lung tissue. The effects of inhibiting Rac1 (using 10
␮M NSC23766) can be seen immediately following drug
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application in this recording; Fig. 7B enlarges channel activity
from the control recording period as well as during NSC23766
treatment to show details (see figure legend). In Fig. 7C, we
show a summary of results from 6 independent patch-clamp
recordings, both before and after Rac inhibition. On average,
inhibition of Rac1 in type 1 cells significantly decreased ENaC
NPo values from 1.16 ⫾ 0.27 to 0.38 ⫾ 0.10; P ⬍ 0.05. Rac
inhibitor (1 ␮M) caused a similar decrease in ENaC activity in
type 2 cells. Again, in Fig. 8A, we show a representative
continuous recording of a cell-attached patch of a type 2 cell
accessed from a lung slice preparation, with portions enlarged
in Fig. 8B to highlight single-channel characteristics. Figure 8C
shows that in each independent cell observation, ENaC NPo in
type 2 cells decreased from 0.87 ⫾ 0.16 to 0.52 ⫾ 0.16 (mean
NPo values ⫾ SE, n ⫽ 6; P ⬍ 0.05) immediately following Rac
inhibition.
DHE measurements of O⫺
2 verify that NSC23766 inhibition
indeed decreases O⫺
2 release in sodium transporting epithelia
(Fig. 9). As we have alluded to above, the reaction product of
⫹
O⫺
2 with DHE yields a fluorescent product, 2-OH-E , and is
measureable both by its elution time in HPLC and its fluorescent properties (excitation 520 nm/emission 610 nm) using a
monochromatic plate reader. Importantly, 2-OH-E⫹ is a product generated specifically by O⫺
2 oxidation of DHE, whereas
ethidium formation is attributed primary to pathways involving
H2O2 (19). In Fig. 9, left, we assessed 2-OH-E⫹ levels of A6
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Fig. 4. Type 1 cells robustly express Rac1
and NOX2. A: equal number FACS-sorted
type 1 (lane 1) and type 2 (lane 2) cells were
lysed and analyzed. Blots show robust Rac1
and NOX2 subunit expression in type 1
cells; NOX2 and Rac1 expression levels in
equal number type 2 cells were not as pronounced. B: relative luminescence intensities from 3 independent experiments (with
protein lysate derived from 3 different animals and FACS); type 2 (AT2) cell values
were set to an arbitrary value of 1, and type
1 (AT1) protein was expressed as fold increase above (X) 1.
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Fig. 5. Immunohistochemical detection of Rac1 and NOX2 in
paraformaldehyde-fixed lung slices. In each figure, the left and
right are of the same cell. The left shows bright field illumination, and right shows fluorescent excitation of antibodies/
vital dyes as described below. All images were original ⫻40
magnification using 200-␮m lung tissue slices. A: right: type 1
cells identified using ECL (green). Anti-rabbit Rac1 antibody
labeling [detected using rabbit secondary antibody conjugated
to Alexa Fluor 568 (red)] shows cytoplasmic Rac1 expression
in type 1 cells. B: right: ECL labeling of type 1 cells, lung slice
counter-stained with anti-rabbit NOX2 antibody (detected using secondary antibody conjugated to Alexa Fluor 568) shows
NOX2 catalytic domain facing lumen of airways. C: right:
type 2 cells identified using LTR. Anti-rabbit Rac1 antibody
labeling [detected using rabbit secondary antibody conjugated
to Alexa Fluor 488 (pseudocolored cyan)] shows Rac1 expression in alveolar epithelial cells. D: LTR labeling of type 2
cells, lung slice counter-stained with anti-rabbit NOX2 antibody detected using secondary antibody conjugated to Alexa
Fluor 488 (pseudocolored cyan).
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Rac1-MEDIATED NOX REGULATES LUNG ENaC
cells treated with Rac inhibitor and found that inhibiting this
small G protein decreased O⫺
2 levels ⬃7-fold (gray bars). We
can be confident that Rac inhibition does not inadvertently alter
other reactive oxygen species, such as H2O2, since the
ethidium levels remained unchanged ⫾ Rac inhibitor (black
bars). After verifying the specificity of DHE as an O⫺
2 sensor,
we repeated studies using primary lung cells and detected the
Fig. 7. NSC23766, a specific Rac1 inhibitor,
decreases ENaC activity in type 1 cells. A: representative patch-clamp recording obtained from
a type 1 cell accessed in situ. #Point of 10 ␮M
NSC23766 application to continuous patch following control recording period. B: enlarged portion of continuous trace showing 8-pS highly
selective channel (HSC) and 37.5-pS nonselective channel (NSC) present in the same patch of
membrane. Inhibition of Rac1 activity decreased
both NSC and HSC activity in representative
trace. Arrows indicate closed state, and inward
currents are seen as downward deflections from
closed state. C: results of 6 independent observations (HSCs and NSCs included) shown on
dot-plot graphs with y-axis ⫽ ENaC number of
open channels (NPo). In each observation plotted, Rac1 significantly decreased ENaC NPo values; on average, NPo decreased from 1.16 ⫾
0.27 to 0.38 ⫾ 0.10; P ⬍ 0.05.
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Fig. 6. ENaC coimmunoprecipitates with NOX2 catalytic domain. Western blot (WB)
analysis of immunoprecipitated (IP) protein, derived from rat primary alveolar epithelial cells, using either goat anti-␣-ENaC subunit antibody (lane 1) or rabbit anti-NOX2
antibody (lane 2; positive signal control). Detection of NOX2 in IP studies was done
so using standard Western blot protocols and rabbit anti-NOX2 primary antibody. The
typical glycosylation smear for NOX2 [which runs between 90 and 65 kDa (20)] was
observed in both lane 1 and 2.
fluorescent properties of DHE using a microplate reader in Fig. 9,
right,. Once again, we confirmed that NSC23766 inhibition of
Rac1 indeed decreases O⫺
2 in alveolar epithelial cells: relative
light units (an alternative fluorescent measure of 2-OH-E⫹) were
significantly higher in vehicle-treated cells. Rac inhibition
significantly decreased the fluorescent detection of O⫺
2 product.
EGF-stimulated Rac1 acutely regulates lung ENaC. Several
studies have shown that EGF stimulates Rac1 (5, 27) and that
EGF receptor inhibition (using AG1478 compound) significantly decreases Rac1 activity with associated decreases in
reactive oxygen species generation (27). In the present study,
we use EGF to acutely increase Rac1 in lung slices and then
measured single-channel activity. Figures 10 and 11 show that
10 ng/ml EGF increases ENaC NPo values in both type 1 and
2 cells, respectively. Continuous traces of cell-attached patches
before and after drug treatment, with enlarged portions to show
detail, are presented for each cell type (Fig. 10, A and B, and
Fig. 11, A and B). In type 1 cells, ENaC activity significantly
increased from 0.40 ⫾ 0.15 to 0.60 ⫾ 0.23 following EGF
treatment in 7 separate observations with P ⬍ 0.05 (Fig. 10C).
In 10 independent type 2 cell observations, NPo values also
increased, on average, from 0.48 ⫾ 0.21 to 0.91 ⫾ 0.28 (P ⬍
0.05; Fig. 11C).
In Fig. 10, D and E, and Fig. 11, D and E, we further
scrutinized the effect of EGF on NPo separately in both type 1
and 2 cell recordings. In type 1 cells, the significant increase in
sodium channel activity following EGF treatment is largely
attributed to increases in N in the surface membrane; Po is not
altered by EGF treatment in type 1 cells (Fig. 10, D and E).
EGF-treated type 2 cells, however, responded with significant
increases in Po and no change in N measured (Fig. 11, D and E).
These differences in the effect of EGF on N in type 1 cells vs.
Po in type 2 cells allude to differences in the inherent regulatory mechanisms governing sodium readsorption in neighboring type 1 and 2 cells.
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Rac1-MEDIATED NOX REGULATES LUNG ENaC
In Fig. 12, the superoxide dismutase mimetic TEMPO was
used to scavenge endogenous O⫺
2 before EGF treatment and
single-channel analysis. By sequestering endogenous levels of
oxygen radicals before EGF activation, we gain additional insight
into O⫺
2 signaling mechanisms in the lung. These experiments
were performed in both type 1 (Fig. 12A) and type 2 cells (Fig.
12B) and show that O⫺
2 is an important signaling molecule in EGF
activation of ENaCs in the lung. Sequestering O⫺
2 before EGF
treatment in both type 1 and 2 cells prevented EGF activation of
ENaC NPo measured by single-channel patch-clamp recordings.
DISCUSSION
FACS reliably purifies alveolar epithelial cells. Although
type 1 cells make up the vast majority of the surface area in the
alveoli, little is known about the biochemical properties of this
cell type. Advancement in our understanding of type 1 cells,
particularly compared with type 2 cells, has progressed slowly
due to limitations in establishing an appropriate model system
for type 1 cells. In the current study, we physically separated
type 1 cells from all other lung cells using FACS. The flow
cytometer simultaneously separated cells based on fluorescence and size profiles (described in RESULTS); as such, the type
1 cells obtained in this manner are of certain purity and
number. We (17) and others (11) have recently used FACS as
a useful approach to obtaining alveolar epithelial cells. In a
similar approach, Gonzalez et al. (11) fluorescently labeled
type 1 and 2 cells (using anti-RTI40 and RTII70 primary
antibodies and the appropriate fluorescent secondary conju-
Fig. 9. Inhibiting Rac1 activity significantly decreases O⫺
2
production. Left: NSC23766 significantly decreases the production of the O⫺
2 -specific product 2-hydroxyethidium (2OH-E⫹; gray bars) without altering levels of other reactive
oxygen species (black bars); n ⫽ 3 independent observations in model A6 cells; P ⬍ 0.05. Right: fluorescent
detection (relative light units; RLU) of 2-OH-E⫹ in primary
alveolar epithelial cells confirm that NSC23766 compound
significantly decreases O⫺
2 production in pneumocytes; n ⫽
3 independent observations, with sample size of ⱖ12 wells
per experiment. Oxy E, oxyethidium; n.s., not significant;
CTR, control.
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Fig. 8. NSC23766 inhibits ENaC in type 2 cells.
A: representative recording of a type 2 cell accessed in situ. NSC23766 Rac1 inhibitor (1 ␮M)
applied to cell-attached patch (near #) following a
control recording period. B: segment of continuous
recording enlarged to show detail of 28-pS NSC
that decreased in activity immediately following
Rac1 inhibition. Arrow indicates closed state, and
downward deflections represent inward Na currents. C: dot-plot graphs showing ENaC NPo before and after drug treatment. NSC23766 decreased channel activity in each independent observation, on average from 0.87 ⫾ 0.16 to 0.52 ⫾
0.16, n ⫽ 6; P ⬍ 0.05.
Rac1-MEDIATED NOX REGULATES LUNG ENaC
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gated antibodies) to purify alveolar epithelial and Clara cells.
Moreover, Gonzalez et al. (11) showed that 85–90% of type 1
cells obtained using FACS maintained viability, were of essential purity, and proliferated under in vitro culture conditions. In our study, we used Western blot analysis to confirm
that there are not contaminating populations of cells in the
sorted type 1 and 2 samples (Fig. 1). Combined, these studies
show that flow cytometry is an efficient and reproducible
methodology to isolate alveolar epithelial cells of interest.
Coupled with our recent development of a live lung tissue
preparation that allows access to intact type 1 and 2 cells for
single-channel patch-clamp analysis (6), we now have the
appropriate tools to compare the biochemical and biophysical
properties of all native alveolar epithelial cells.
A new role for NOX2 in lung ENaC regulation. To learn
more about O⫺
2 signaling in the lung, we first compared the
level of NOX expression in type 1 and 2 cells to gain a better
understanding of where O⫺
2 signaling would most likely exert
a physiological effect. First, we showed that the NOX2 multiunit complex, and small G protein Rac1, are expressed at
greater levels in type 1 cells compared with neighboring type
2 cells. In our study, Western blot analysis was normalized to
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cell number, since type 1 and 2 cells are not uniform in
thickness nor protein composition. Because type 1 cells are
reportedly only 0.35-␮m thick, and type 2 cells are cuboidal
with a uniform cell thickness of 10 ␮m (yet considerably
smaller than type 1 cells), the protein content of each cell type
must vary significantly (in terms of membrane vs. cytoplasmic
protein expression). Hence, comparison of NOX2 subunit expression between equal number type 1 and 2 cell is most
indicative of the relative contribution each cell type makes to the
immediate redox environment. Based on our Bradford protein
concentration measurements, type 1 cells typically have twice the
protein content of a type 2 cell. Our findings, however, were that
NOX2 subunit and monomeric G protein expression was ⱖ8-fold
higher in type 1 cells compared with type 2 cells. These findings
were consistent in three independent observations of flow-sorted
type 1 and 2 cells performed in parallel. Of particular interest,
however, is that these findings are in line with our recent report
that type 1 cells generate significantly higher amounts of reactive
oxygen species than type 2 cells (12), resulting in different
responses to nitric oxide and ENaC activity. Seemingly, greater
NOX2 expression in the type 1 cells contributes to the overall
oxidative state, and cellular responses, in the alveoli.
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Fig. 10. EGF acutely increases ENaC activity in type 1 cells. A: representative cellattached patch-clamp recording obtained
from type 1 cell accessed in situ. EGF (10
ng/ml) added to patch solution following
control recording period (#). B: portion of
trace from A enlarged to clearly show that
channels spend less time in closed state (indicated by dashed line extending from arrow
pointing to closed state) following EGF
treatment; NSCs with a 22-pS conductance
are shown. C: EGF acutely increased ENaC
NPo values from 0.40 ⫾ 0.15 to 0.60 ⫾ 0.23;
*P ⬍ 0.05; n ⫽ 7. D: the open probability
(Po) of type 1 cells examined did not change
significantly following EGF treatment. E: EGF
increases ENaC NPo via significant changes in
the number of active channels (N) in the
membrane.
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Rac1-MEDIATED NOX REGULATES LUNG ENaC
To confirm the finding that type 1 and 2 cells may differ
significantly in NOX2 release of O⫺
2 , we performed additional
immunohistochemical studies. Figure 5 reveals that NOX2
catalytic subunit is predominantly localized in type 1 cells and
to a lesser extent in type 2 cells. Although we could not
demonstrate colocalization of NOX2 with sodium channels,
co-IP studies circumvented the detection limit of native ENaC
using commercially available antibodies and standard confocal
microscopy. In Fig. 6, we detected NOX2 from ␣-ENaC-bound
immune complexes (i.e., ENaC subunits were immunoprecipi-
Fig. 12. Sequestering O⫺
2 abrogates EGFinduced changes in ENaC activity. In continuous patch-clamp recordings, type 1 (A)
and type 2 cells (B) were treated with 10
ng/ml EGF after 250 ␮M 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) treatment. Sequestering O⫺
2 in type 1 cells significantly decreased ENaC activity [from
0.40 ⫾ 0.18 to 0.06 ⫾ 0.05 (in the presence
of EGF); P ⫽ 0.03; n ⫽ 3] and also prevented EGF-induced increases. TEMPOtreated type 2 cells did not respond significantly to EGF treatment in 10 independent
observations.
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Fig. 11. Acute increases in ENaC following
EGF treatment in type 2 cells. A: representative cell-attached patch-clamp recording of
type 2 cell accessed in situ. EGF was added
to bath media (as indicated near # in continuous patch), which led to immediate increases in channel activity. B: enlarged portions of continuous trace to show detail of
channels under control and EGF treatment;
representative channel shown has a 10-pS
conductance. C: in type 2 cells, ENaC NPo
values significantly increased from 0.48 ⫾
0.21 to 0.91 ⫾ 0.28 (P ⬍ 0.05; n ⫽ 10)
following EGF stimulation. D: EGF significantly altered the Po of type 2 cells examined and had no effect on the number of
active channels in the cell membrane (E).
Rac1-MEDIATED NOX REGULATES LUNG ENaC
AJP-Lung Cell Mol Physiol • VOL
centration of NSC23766 needed to block ENaC function in
type 1 and 2 cells require further investigation.
Conversely, EGF (a known stimulator of both Rac1 and O⫺
2)
led to significant increases in ENaC activity in both type 1 and
2 cells, albeit via different mechanisms. In general, increases in
sodium channel activity can be attributed to either N and/or Po.
We report in Fig. 10, D and E, that the 50% increase in sodium
transport following EGF stimulation in type 1 cells is due to an
increase in the number of active channels. However, EGF
stimulation of type 2 cell activity (from an average NPo value
of 0.48 ⫾ 0.21 to 0.91 ⫾ 0.28; Fig. 11, D and E) is due to
significant changes in the Po of channels residing in the
membrane. Based on our current finding that primary type 1
cells have a greater propensity for releasing O⫺
2 (because they
express greater levels of NOX) over type 2 cells and our
previous report (12) that cultured type 1 cells release more O⫺
2
than type 2 cells, we speculate that the amount of O⫺
2 release
may be accountable for the differences in N and Po effect.
Seemingly, high levels of O⫺
2 release in type 1 cells led to
significant increases in the number of active channels in the
cell membrane (as shown in Fig. 10E). Perhaps the large
NOX-mediated increase in O⫺
2 release stabilizes sodium channel subunits in the membrane. This is a plausible explanation to
investigate further, given that oxidation of cysteine residues
form disulfide bridges that can act to stabilize proteins. Each
ENaC subunit, indeed, expresses conserved cysteine residues
in the large extracellular loops of the ␣-, ␤-, and ␥-subunits (9).
In type 2 cells, however, changes in the gating properties (i.e.,
Po) were observed following EGF treatment (Fig. 11). In type
2 cells, perhaps low levels of O⫺
2 release indirectly modulate
regulatory proteins that have known effects on regulating
ENaC Po (reviewed and discussed in Refs. 23, 24, 26). Additional evidence for direct O⫺
2 regulation of ENaC in type 1
cells, as opposed to indirect redox signaling in type 2 cells, can
be gleaned from results in Fig. 12. In Fig. 12, sequestering
endogenous O⫺
2 levels led to significant decreases in ENaC
activity within minutes, even in the presence of EGF stimulation, in type 1 cells only. This observation supports the contention that O⫺
2 signaling is necessary and required for direct
sodium channel activity in type 1 cells, whereas type 2 cells
have compensatory and/or indirect ENaC regulatory pathways
that are not redox-sensitive (TEMPO and EGF cotreatment
neither increased nor decreased sodium current in type 2 cells).
Given that the redox state of the alveolar microenvironment
can change quickly and drastically from moment to moment,
having multiple regulatory pathways controlling net salt and
water balance is crucial for maintaining normal lung function.
In summary, we show that robust expression of Rac1 and
NOX2 subunits in type 1 cells accounts for the higher oxidative
state that these cells exist in, compared with type 2 cells. Singlechannel analysis of intact alveoli indicates that Rac1-mediated
NOX2 release of O⫺
2 plays an important role in regulating normal
sodium channel activity. Absent Rac1 activation of NOX2, ENaC
activity is significantly decreased in both type 1 and 2 cells.
Conversely, immediate activation of Rac1 by EGF led to associated increases in ENaC activity measured by patch-clamp analysis. This study also indicates that small G protein signaling (Rac1)
and regulated NOX release of O⫺
2 cross talks with signals that
ultimately merge to regulate ENaC activity. Although the study
makes evident the physiological role that NOX2 plays in maintaining lung fluid homeostasis via impacting sodium channel
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tated and subsequently subjected to Western blot analysis using
anti-NOX2 antibody). The implication is that O⫺
2 molecules
released by NOX2 could act locally and immediately on ENaC
before reactive species become inactivated by antioxidants.
This is an important consideration, given that reactive species
are notorious for short half-lives and quick reactivity. It is
important to note that we performed a related study in which
NOX2 protein was immunoprecipitated from lung cell lysate
and subsequently subjected to Western blot analysis using
anti-␣-ENaC antibody (data not shown). When IP-ing with
NOX2 antibody, ␣-ENaC could not be detected from the
NOX2-bound immune complexes. One simple interpretation is
that the ␣-ENaC antibody works effectively to pull-down
sodium channel subunits from cell lysate but may be somewhat
less efficient in detecting protein in Western blots. Alternatively, however, we can make inferences to the presence and
quantity of protein-protein interactions between ENaC and
NOX2 in the lung. Given that NOX2 could be detected from
␣-ENaC immune complexes using standard Western blot (Fig. 6),
we can infer that a large proportion of ␣-ENaC subunits
associate with NOX2 in lung cells (and NOX2 is hence easily
detected in Western blot). Conversely, however, given that
␣-ENaC was not detected from NOX2-immunoprecipitated
protein complexes, it may be the case that of all the NOX2
pulled down from lung cell lysate, a smaller percentage of
NOX2 associates with ENaC (compared with the percentage of
ENaCs that associate with NOX2). ENaC is therefore undetected in Western blot analysis when the NOX2 immune
complex is transferred to nitrocellulose membrane and probed
with anti-␣-ENaC antibody (data not shown).
Using single-channel patch analysis and access to type 1 and
2 cells via live tissue slice preparations, we determined the
acute effects of activating or inhibiting small Rac1 GTPase
activity (and hence, subsequent NOX2 function). We examined
the immediate effects of Rac1-mediated O⫺
2 signaling and its
impact on sodium channel function, since the immediate early
effect of O⫺
2 release on ENaC activity (within minutes) may be
the most important time point to scrutinize in single-channel
recordings given that reactive oxygen species can be quickly
inactivated by antioxidants. Inhibition of Rac1, using
NSC23766, led to significant decreases in ENaC activity in
both type 1 and 2 cells that were detectable within ⬃5 min of
treatment. NSC23766 has been characterized as a specific Rac1
inhibitor (30) that works by competing with guanine exchange
factor (GEF) binding in the surface groove (centering Trp56) of
Rac1. In our studies, we found that 1 ␮M Rac1 inhibitor
effectively turned off ENaC activity in type 2 cells (Fig. 8),
whereas 10 ␮M NSC23766 was needed to observe a significant
decrease in ENaC activity in type 1 cells. This may be an
important observation to further investigate, as it may reveal
additional fundamental differences in the two cell types that
make up the alveoli. Because Rac1 is regulated, in part, by
GEFs that catalyze nucleotide exchange, the observed differences in effective NSC23766 concentrations in Fig. 8 may
possibly be due to differences in GEFs or GEF action in each
cell type examined. Alternatively, activated Rac1 may bind to
different downstream target proteins in type 1 and 2 cells that
may require different concentrations of Rac1 inhibitor to elicit
the same inhibitory effect on sodium channel activity. Again,
these speculative explanations for the different effective con-
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Rac1-MEDIATED NOX REGULATES LUNG ENaC
ACKNOWLEDGMENTS
We acknowledge the technical assistance of Julie L. Self in Western blot
procedures.
GRANTS
This work was supported by National Institutes of Health Grant K99-HL09222601 awarded to M. N. Helms. This research was also supported, in part,
by the Parker B. Francis Foundation, Emory University’s Research Committee
Grant, and the Flow Cytometry Core Facility of the Emory University School
of Medicine.
DISCLOSURES
No conflicts of interest are declared by the author(s).
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regulation, the precise molecular mechanism by which O⫺
2 could
regulate the immediate increase in ENaC activity requires further
investigation. Although speculative, we allude above to the possibility that oxidized cysteines on sodium channel subunits may
form disulfide linkages that would increase the number of active
channels in type 1 membrane.
CFTR and correlations with O⫺
2 regulation of ENaC. The
interrelatedness of the cystic fibrosis transmembrane regulator
(CFTR) and normal ENaC regulation (reviewed in Ref. 2) is
evident in cystic fibrosis lung disease. Interestingly, CFTR activity influences the oxidative state of the cell. Specifically, wild-type
CFTR conducts glutathione (21). As such, cells with mutations in
CFTR would have a decrease in the ability to secrete glutathione
and buffer antioxidants in cells. The resultant increase in reactive
oxygen species could account for the observed increase in salt
readsorption in cystic fibrosis disease. Our experimental evidence
supports the notion that the rise in radical oxygen species, caused
by a decrease in glutathione buffering, could contribute to inappropriate sodium channel activation observed in CFTR. A better
understanding of redox regulation of lung ENaC can lead to
possible therapeutic insight into CFTR lung disease.