Am J Physiol Lung Cell Mol Physiol
281: L575–L581, 2001.
Chronic ethanol downregulates PKA activation and
ciliary beating in bovine bronchial epithelial cells
T. A. WYATT AND J. H. SISSON
Research Service, Department of Veterans Affairs Medical Center, Omaha 68105;
and Pulmonary and Critical Care Medicine Section, Department of Internal
Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198
Received 31 July 2000; accepted in final form 3 April 2001
lung; airway; adenosine 3⬘,5⬘-cyclic monophosphate; downregulation; ␤-agonist; cyclic nucleotide; cAMP-dependent
protein kinase
CLINICAL STUDIES have demonstrated that airway host
defenses are impaired in alcoholics (6, 17). Chronic
alcohol consumption is associated with a high incidence of bronchitis, pneumonia, and aspiration (2, 15).
The mucociliary escalator serves as the frontline defense for the lung against inhaled microorganisms,
particles, and debris. This regulatable host defense is
likely impaired by the excessive consumption of alcohol
and contributes to the increased risk and presentation
of lung disease in alcoholics. The exact mechanisms of
impaired mucociliary function in response to ethanol
consumption remain poorly understood.
Changes in mucociliary transport can be experimentally modeled by monitoring changes in ciliary beat
frequency (CBF). CBF is likely increased during con-
Address for reprint requests and other correspondence: T. A.
Wyatt, Dept. of Internal Medicine, Pulmonary and Critical Care
Medicine Section, Univ. of Nebraska Medical Center, 985300
Nebraska Medical Center, Omaha, NE 68198-5300 (E-mail:
[email protected]).
http://www.ajplung.org
ditions of airway epithelial cell “stress.” This can occur
transiently as part of the “fight or flight” response to
substances such as ␤-agonists, substance P, and bradykinin (10, 20, 35, 39). Alternatively, ciliary stimulation can be prolonged in response to inflammation via
cytokines such as tumor necrosis factor-␣ or interleukin (IL)-1␤ released from inflammatory cells (9). Previously, we have studied the acute stimulation of CBF
by demonstrating that agents that increase either
cGMP or cAMP and subsequently activate either
cGMP-dependent protein kinase (PKG) or cAMP-dependent protein kinase (PKA) lead to increased CBF in
the bovine ciliated airway epithelial cell (39). We have
also demonstrated that ethanol rapidly and transiently
stimulates airway epithelial cell CBF in vitro and that
a nitric oxide (NO)-dependent mechanism is involved
in these ethanol-stimulated CBF increases (31, 32).
Additionally, there appears to be a cAMP-regulatable
component to acute ethanol-stimulated CBF increases
as ethanol activates PKA in the airway epithelial cell
(32). Thus we have found that ethanol signaling of CBF
is directly linked to the activation of PKA and is indirectly linked to activation of PKG.
The rapid and transient stimulatory effects of acute
ethanol exposure on ciliary motility in vitro are seemingly inconsistent with the impaired airway host defenses that ethanol is known to cause in alcoholics.
Because independently regulated NO-dependent mechanisms have been described for acute vs. chronic activation of ciliary motility (9), we explored the long-term
responses of airway cell CBF to ethanol. Because impaired host defenses are associated with chronic ethanol consumption, we hypothesize that chronic exposure
to ethanol blunts NO-dependent ciliary stimulation
through downregulation of PKA. This downregulation
of the mechanism by which CBF is increased correlates
with the ethanol-associated impairment in mucociliary
clearance.
In this study, we show that the early stimulation in
ciliary activity observed after acute ethanol treatment
is followed by a return to baseline (unstimulated) CBF
between 6 and 24 h in bovine bronchial epithelial cells
The costs of publication of this article were defrayed in part by the
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solely to indicate this fact.
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Wyatt, T. A., and J. H. Sisson. Chronic ethanol downregulates PKA activation and ciliary beating in bovine bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol
281: L575–L581, 2001.—Previously, we reported that ethanol (EtOH) stimulates a rapid increase in ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBEC).
Agents activating cAMP-dependent protein kinase (PKA)
also stimulate CBF. EtOH stimulates BBEC CBF through
cyclic nucleotide kinase activation. However, EtOH-stimulated CBF is maximal by 1 h and subsides by 6 h, returning
to baseline by 24 h. We hypothesized that the loss of EtOHstimulated CBF was a result of downregulation of PKA
activity. To determine the PKA activation state in response
to EtOH, ciliated BBEC were stimulated for 0–72 h with
various concentrations of EtOH and assayed for PKA. EtOH
(100 mM) treatment of the cells for 1 h increased PKA
activity threefold over unstimulated controls. PKA activity
decreased with increasing time from 6 to 24 h. When BBEC
were preincubated with 100 mM EtOH for 24 h, the stimulation of PKA by isoproterenol or 8-bromo-cAMP was abrogated. EtOH desensitizes BBEC to PKA-activating agents,
suggesting that EtOH rapidly stimulates, whereas long-term
EtOH downregulates, CBF via PKA in BBEC.
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CHRONIC ETHANOL DESENSITIZES CILIA
(BBEC). Similarly, ethanol-stimulated PKA activity
also declines to unstimulated levels in a parallel manner. We have observed that the BBEC become desensitized to further ethanol-stimulated increases in CBF
and PKA at these later time points. Importantly, after
long-term ethanol treatment, BBEC are desensitized
to additional CBF increases because of agents that
otherwise acutely stimulate CBF. Our observations
suggest that chronic ethanol exposure of BBEC results
in the desensitization of PKA-mediated CBF.
MATERIALS AND METHODS
AJP-Lung Cell Mol Physiol • VOL
RESULTS
Ethanol-stimulated PKA activity decreases over time.
To determine the duration of ethanol-stimulated PKA
activity in airway epithelial cells, crude homogenates
of BBEC were fractionated, and the soluble cytosolic
fraction was assayed for PKA (see MATERIALS AND METHODS). BBEC treated with 10–100 mM ethanol for ⬃1 h
demonstrate maximal activation of PKA (Fig. 1). This
activity begins to diminish between 2 and 6 h, returning to baseline (unstimulated) PKA activity after 6–8
h. Concentrations of ethanol ⬍10 mM fail to activate
PKA. After ethanol-stimulated PKA activity returned
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Cell preparation. As previously described (36), the cells
were prepared from bovine lung obtained fresh from a local
abattoir. Bronchi were necropsied from the lung, cleaned of
adjoining lung tissue, and incubated overnight at 4°C in 0.1%
bacterial protease (type IV) in MEM. After overnight incubation, the bronchi were rinsed in DMEM with 10% FCS repeatedly to collect the cells lining the lumen. This technique
typically produces a high-viability cell preparation of ⬎95%
epithelial cells (29). The cells were then washed in DMEM,
counted with a hemacytometer, and plated in 1% collagencoated 100-mm polystyrene petri dishes at a density of 1 ⫻
104 cells/cm2 in a 1:1 medium mixture of LHC-9 and RPMI
(16). Cell incubations were performed at 37°C in humidified
95% air-5% CO2. Confluent monolayers of cells were obtained
every 3 days. At this time, each 60-mm dish contained ⬃2 mg
of total cellular protein. Primary cultures of BBEC were used
for these studies, as it has been suggested that tissue culture
artifact may induce the downregulation of certain enzyme
activity in the late-passaged cell (3).
Determination of cyclic nucleotide-dependent kinase activity. PKA activity was determined in crude whole cell fractions of BBEC. The assay employed is a modification of
procedures previously described (11) using 130 ␮M PKA
substrate heptapeptide (LRRASLG), 10 ␮M cAMP, 0.2 mM
IBMX, 20 mM magnesium acetate, and 0.2 mM [␥-32P]ATP in
a 40 mM Tris 䡠 HCl buffer (pH 7.5). Samples (20 ␮l) were
added to 50 ␮l of the above reaction mixture and incubated
for 15 min at 30°C. Spotting 50 ␮l of each sample onto P-81
phosphocellulose papers halted incubations. Papers were
then washed five times for 5 min each in phosphoric acid (75
mM), washed one time in ethanol, dried, and counted in
nonaqueous scintillant as previously described (25). Negative
controls consisted of similar assay samples with or without
the appropriate substrate peptide or cyclic nucleotide. A
positive control of 0.4 ng/ml purified catalytic subunit from
type I bovine PKA (Promega) was included as a sample.
Kinase activity is expressed in relation to total cellular protein assayed and was calculated in picomoles per minute per
milligram. All samples were assayed in triplicate, and no less
than three separate experiments were performed per unique
parameter. Data were analyzed for statistical significance
using Student’s paired t-test.
Determination of cyclic nucleotide levels. Cyclic nucleotide
levels were determined using a protein kinase activation
assay as previously described (39). Briefly, cell monolayers
were flash frozen in liquid N2 after addition of 1 ml KPEM
(10 mM KH2PO4, 1 mM EDTA, and 25 mM 2-mercaptoethanol) per dish. Cell protein extracts were transferred to microfuge tubes and boiled at 95°C for 5 min. After centrifugation (10,000 g for 30 min), the supernatants were diluted 1:10
in 10 mM potassium phosphate buffer (pH 6.8) containing 0.9
mg/ml BSA, and 20 ␮l were added to 50 ␮l of stock reaction
mixture consisting of 40 mM Tris 䡠 HCl (pH 7.4), 20 mM
magnesium acetate, 130 ␮M kemptide (LRRASLG), and 0.2
mM [␥-32P]ATP. Reactions were initiated by the addition of
10 ␮l of PKA (24) diluted to 0.4 nM with KPEM and 0.9
mg/ml BSA. After incubation at 4°C for 16–20 h, 50-␮l
aliquots were spotted on phosphocellulose paper (Whatman
P-81) and placed immediately in 75 mM phosphoric acid. The
papers were then washed, dried, and counted in nonaqueous
scintillant (25). The assay for cGMP levels was performed
similarly as above, substituting partially purified PKG (18),
and 150 ␮M heptapeptide substrate (RKRSRAE) specific for
PKG was substituted for kemptide. PKA inhibitor peptide
(15 ␮M) was also added to the reaction mixture. All incubations were performed in duplicate, and each experiment was
repeated three or more times. Cyclic nucleotide concentrations (pmol/mg protein) were determined by comparison with
a standard curve of cyclic nucleotide-activated kinase activities (pmol 䡠 min⫺1 䡠 ml⫺1) that was performed concurrently
with each experiment. Protein in each sample was measured
by the technique of Bradford (1) and was used to standardize
for each experiment.
CBF measurements. Actively beating ciliated cells were
observed, and their motion was quantified by measuring CBF
using phase-contrast microscopy, videotape analysis, and
computerized frequency spectrum analysis. Ciliated cells in
culture were maintained at a constant temperature (24 ⫾
0.5°C) by a thermostatically controlled heated stage. The
cells were maintained at room temperature during the time
course of the CBF measurements, since the temperature
gradient was known to affect CBF (27). All observations were
recorded for analysis using a Panasonic WV-D5000 video
camera and a Panasonic AG-1950 videotape recorder. Beat
frequency analysis was performed on videotaped experiments using customized software written in LabView (National Instruments, Austin, TX) running on a Macintosh G3
computer. The predominant frequency of a cilium or small
group of cilia was determined by collecting data sampled at
40 Hz from 512 samples (12.8 s) and performing frequency
spectrum analysis. The CBF determined in this manner was
deemed acceptable when a single dominant frequency was
obtained using this technique. All frequencies represent
means ⫾ SE from six separate cell groups or fields.
Materials. LHC basal medium was purchased from Biofluids (Rockville, MD). RPMI 1640 medium, DMEM, MEM,
streptomycin-penicillin, and Fungizone were purchased from
GIBCO BRL (Chagrin Falls, OH). Extraction of frozen bovine
pituitaries from Pel Freez (Rogers, AR) was performed as
previously described and yielded an extract containing 10
mg/ml protein (16). [␥32-P]ATP was from Amersham, phosphocellulose P-81 paper was from Whatman, peptide substrates
were from Peninsula Laboratories, and absolute ethanol was
from McCormick Distilleries. All other reagents not specified
were purchased from Sigma Chemical (St. Louis, MO).
CHRONIC ETHANOL DESENSITIZES CILIA
to baseline levels, no further increase in PKA was
observed from 18 to 64 h (data not shown). The cell
medium was then exchanged each hour with a fresh dose
of 100 mM ethanol in new medium so that the concentration of ethanol across all time points would remain
constant at 100 mM. With the use of this medium replacement technique, the pattern of PKA activation
was again observed to return from maximal activation
back to baseline levels between 6 and 8 h (Fig. 2). This
suggests that ethanol is not being consumed by metabolism or volatilization under our experimental condi-
Fig. 2. Time course of PKA activation in BBEC treated with repeated doses of fresh EtOH. Monolayers of BBEC were treated with
fresh medium containing either 100 mM EtOH or control medium
every hour from 1 to 8 h, and PKA activity was assayed. Each data
point represents the average of triplicate measurements of 3 or more
samples within an experiment (P ⱕ 0.05 for EtOH-stimulated PKA
activity compared with cells treated with control medium from 1 to
6 h. No significant difference exists between conditions at 7 and 8 h).
AJP-Lung Cell Mol Physiol • VOL
tions. These data indicate that prolonged exposure to
ethanol results in a downregulation or autoinactivation of PKA activity to continued ethanol exposure.
Ethanol-stimulated CBF decreases over time. To determine the duration of ethanol-stimulated increases
in CBF in airway epithelial cells, ciliated primary
cultures of BBEC were treated with 100 mM ethanol,
and ciliary motility was measured (see MATERIALS AND
METHODS). With no pretreatment, ethanol stimulates
CBF by 1 h with a return to baseline levels after 6 h
(Fig. 3). When the cells were pretreated with 100 mM
ethanol for 24 h and then reexposed to fresh medium
containing 100 mM ethanol, no significant increase in
CBF was observed, similar to BBEC exposed to control
medium. This suggests that prolonged exposure to ethanol (ⱖ6 h) causes desensitization of ciliary motility to
ethanol and that long-term exposure to ethanol inhibits increases in CBF as a result of subsequent ethanol
challenges.
Ethanol desensitizes BBEC to other CBF stimuli. To
determine if the desensitization of ethanol-stimulated
PKA activity in BBEC affects further stimulation of
CBF by activators of PKA, cells pretreated with 100
mM ethanol (or medium) for 24 h were then exposed to
100 ␮M isoproterenol or 10 ␮M 8-bromo-cAMP (8BrcAMP). BBEC pretreated with medium demonstrated
significant elevations in CBF upon treatment with
either isoproterenol or 8-BrcAMP (Fig. 4B). In sharp
Fig. 3. Effects of long-term EtOH exposure on ciliary beat frequency
(CBF) stimulation (stim) in BBEC. Monolayers of BBEC were pretreated (pretreat) for 24 h with either 100 mM EtOH or control
medium followed by treatment with 100 mM EtOH or control medium from 0 to 6 h, and CBF was assayed. CBF was assayed as
described (see MATERIALS AND METHODS) using videomicroscopy and
frequency spectrum analysis. Each data point represents the average of triplicate measurements of 3 or more samples within an
experiment [P ⱕ 0.05 for cells pretreated with medium and stimulated with 100 mM EtOH for 1, 2, and 4 h (*) compared with cells
treated with medium only or pretreated with EtOH for 24 h]. t, Time.
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Fig. 1. Time course of cAMP-dependent protein kinase (PKA) activation in ethanol (EtOH)-treated bovine bronchial epithelial cells
(BBEC). Monolayers of BBEC were treated with 0.01–100 mM EtOH
from 0 to 16 h, and PKA activity was assayed. Kinase assays were
performed as described (see MATERIALS AND METHODS) using specific
heptapeptide substrates for PKA. Each data point represents the
average of triplicate measurements of 3 or more samples within an
experiment [P ⱕ 0.05 for 10 and 100 mM EtOH-stimulated PKA
activity (*) compared with cells treated with medium only at 1, 2, and
6 h]. Vertical bars represent ⫾SE in Figs. 1–6.
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CHRONIC ETHANOL DESENSITIZES CILIA
contrast, pretreatment of cells with 100 mM ethanol
inhibited any increases in CBF resulting from isoproterenol or 8-BrcAMP (Fig. 4A). Interestingly, a prolonged exposure to 100 mM ethanol also inhibited the
ability of 10 ␮M sodium nitroprusside (SNP) or 10 ␮M
8-bromo-cGMP (8-BrcGMP) to stimulate CBF in
BBEC. These findings parallel the effects of chronic
ethanol exposure on PKA activity (Fig. 5). BBEC pretreated with medium demonstrated significant increases in PKA activity upon treatment with either
isoproterenol or 8-BrcAMP (Fig. 5A). However, pretreatment of cells with 100 mM ethanol inhibited any
significant increases in PKA as a result of isoproterenol
Fig. 5. Effects of long-term EtOH exposure on agonist-stimulated
PKA activity in BBEC. Monolayers of BBEC were pretreated for 24 h
with either 100 mM EtOH (B) or control medium (A) followed by
treatment with 10 ␮M 8-BrcAMP (cAMP), 10 ␮M 8-BrcGMP (cGMP),
100 ␮M isoproterenol, 10 ␮M sodium nitroprusside, or control medium from 2 to 4 h, and PKA activity was assayed. Each data point
represents the average of triplicate measurements of 3 or more
samples within an experiment (P ⱕ 0.05 for cells pretreated with
medium and stimulated with cAMP or isoproterenol for 2–4 h compared with cells treated with medium only or cells pretreated with
EtOH for 24 h followed by agonist stimulation).
AJP-Lung Cell Mol Physiol • VOL
or 8-BrcAMP (Fig. 5B). As expected, neither 10 ␮M
SNP nor 10 ␮M 8-BrcGMP stimulates PKA activity in
BBEC.
Although the use of a phosphodiesterase-resistant
cyclic nucleotide analog to directly activate PKA circumvents the problem of phosphodiesterase activation
or adenylyl cyclase inactivation, we have observed the
same chronic ethanol desensitization of forskolin-stimulated PKA in our system, suggesting that cellular
impermeability to 8-BrcAMP is not occurring (Fig. 6).
As depicted in Table 1, no decreases in isoproterenol-,
forskolin-, or 8-BrcAMP-stimulated cAMP levels were
detected in response to ethanol at any time point observed. Elevations in cAMP were consistently detected
after 1–2 h of treatment with 100 mM ethanol, with a
subsequent return to baseline levels as previously re-
Fig. 6. Effects of long-term EtOH exposure on forskolin (FSK)stimulated PKA activity in BBEC. Monolayers of BBEC were pretreated for various times (0, 1, 6, or 24 h) with 100 mM EtOH
followed by treatment with 100 ␮M forskolin for 30 min, and PKA
activity was assayed. Each data point represents the average of
triplicate measurements of 3 or more samples within an experiment
(P ⱕ 0.05 for cells pretreated with medium or 1 h EtOH and
stimulated with forskolin for 30 min compared with cells pretreated
with EtOH for 24 h followed by agonist stimulation).
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Fig. 4. Effects of long-term EtOH exposure on agonist-stimulated CBF in BBEC.
Monolayers of BBEC were pretreated for
24 h with either 100 mM EtOH (A) or
control medium (B) followed by treatment
with 10 ␮M 8-bromo-cAMP (8-BrcAMP),
10 ␮M 8-bromo-cGMP (8-BrcGMP), 100
␮M isoproterenol (Iso), 10 ␮M sodium nitroprusside (SNP), or control medium
from 0 to 6 h, and CBF was assayed. Each
data point represents the average of triplicate measurements of 3 or more samples
within an experiment (P ⱕ 0.05 for cells
pretreated with medium and stimulated
with agonists for 1–6 h compared with
cells treated with medium only or cells
pretreated with EtOH for 24 h followed by
agonist stimulation).
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CHRONIC ETHANOL DESENSITIZES CILIA
Table 1. Ethanol effects on isoproterenol, forskolin, 8-BrcAMP, SNP, and 8-BrcGMP
Agonist, 1 h
Medium
Isoproterenol (100 ␮M)
Forskolin (100 ␮M)
8-BrcAMP (10 ␮M)
SNP (10 ␮M)
8-BrcGMP (10 ␮M)
Preincubation With 100 mM Ethanol, h
Cyclic Nucleotide,
pM/mg protein
0
1
6
24
cAMP
cGMP
cAMP
cGMP
cAMP
cGMP
cAMP
cGMP
cAMP
cGMP
cAMP
cGMP
0.78 ⫾ 0.12
0.35 ⫾ 0.1
6.2 ⫾ 0.85
1.4 ⫾ 0.3
8.4 ⫾ 1.3
0.92 ⫾ 0.4
11.2 ⫾ 0.9
0.38 ⫾ 0.5
1.6 ⫾ 0.2
4.1 ⫾ 0.6
1.1 ⫾ 0.3
8.6 ⫾ 1.3
4.8 ⫾ 0.3
0.91 ⫾ 0.2
7.2 ⫾ 0.6
1.1 ⫾ 0.3
7.9 ⫾ 1.4
1.1 ⫾ 0.3
10.2 ⫾ 2.2
0.84 ⫾ 0.9
4.1 ⫾ 1.1
5.2 ⫾ 0.9
4.5 ⫾ 0.4
8.5 ⫾ 1.8
1.1 ⫾ 0.3
0.32 ⫾ 0.3
6.6 ⫾ 0.5
0.9 ⫾ 0.3
8.4 ⫾ 0.4
0.96 ⫾ 0.3
13.3 ⫾ 1.7
0.52 ⫾ 0.2
1.3 ⫾ 0.5
3.9 ⫾ 0.1
1.1 ⫾ 0.5
7.9 ⫾ 3.1
1.2 ⫾ 0.2
0.28 ⫾ 0.2
6.3 ⫾ 0.3
1.2 ⫾ 0.2
8.3 ⫾ 1.6
0.71 ⫾ 0.3
13.7 ⫾ 0.9
0.28 ⫾ 0.1
1.3 ⫾ 0.2
4.1 ⫾ 0.1
1.5 ⫾ 0.3
8.3 ⫾ 2.2
ported (32). Ethanol alone did not alter the SNP- or
8-BrcGMP-stimulated levels of cGMP (Table 1). We
also did not detect any decrease in BBEC baseline PKG
activity resulting from chronic ethanol exposure (data
not shown). These data indicate that a prolonged exposure to ethanol blunts airway epithelial cell responsiveness to additional challenges by agents that stimulate CBF increases in vitro.
DISCUSSION
In contrast to our earlier observations with acute
ethanol exposure (31), the current findings suggest
that chronic exposure of ciliated cells to ethanol results
not only in a loss of the acute cilia stimulation effect
but also desensitization of the ciliated cell to subsequent stimulation by ethanol. Not only does ethanol
desensitize the ciliated cell to subsequent ethanol stimulation, but it also blocks stimulation by ␤-agonists.
These observations indicate that chronic exposure to
ethanol impairs the ciliated cell’s ability to increase
motility, as might be required during infection or exercise. Closely associated with cilia desensitization,
chronic ethanol exposure appears to downregulate
PKA, suggesting a possible mechanism for cilia desensitization.
The tolerance of human tissues to high concentrations of ethanol is a peculiar and established quality of
this compound. As we have previously reported (31,
32), the concentrations of ethanol required for CBF
stimulation in BBEC correspond to “legal intoxication”
levels (⬃25 mM), with the maximum increases in CBF
occurring at 100 mM. The concentration of ethanol
used in this study (100 mM) has been demonstrated to
be pathophysiologically relevant to the heavy consumption of alcohol by humans (14). This concentration is equivalent to the blood alcohol content of an
individual who is legally intoxicated (8). Indeed, an
established animal model of chronic alcohol disease,
the Tsukamoto-French diet (5), uses levels of ethanol
well beyond 100 mM for up to 8 wk before levels of
toxicity are observed. This would suggest that the
desensitization of PKA-mediated ethanol stimulation
of CBF is not the result of cellular toxicity.
AJP-Lung Cell Mol Physiol • VOL
To control for a global toxicity effect of ethanol on the
BBEC, we have assayed numerous cell functions after
100 mM ethanol treatment. Our cell viability studies
(lactate dehydrogenase release and trypan blue exclusion) show that 100 mM ethanol is not cytotoxic to the
cells. Additionally, the protein kinase C-mediated release of IL-8 in these cells (37, 38) remains intact after
ethanol washout, further supporting the fact that
chronic ethanol treatment specifically desensitizes
CBF (data not shown). Also, the baseline levels of CBF
continued throughout extended ethanol treatment and
did not decrease below medium-conditioned baseline
CBF levels, as would be expected in a dying cell. Only
the “flight response” as induced by agonist stimulation
of cilia was affected by chronic treatment with ethanol.
Salathe and Bookman (26) have suggested that this
“idle speed” of cilia is regulated by calcium waves, not
by cyclic nucleotides. Furthermore, 100 mM ethanoltreated BBEC can continue to proliferate and migrate
beyond 24 h to close a wound in the in vitro woundhealing model of Kim et al. (13), normal cell functions
that would not occur in the toxic dying cell. Finally,
cyclic nucleotide measurements demonstrate that adenylyl cyclase continues to be stimulatable after longterm ethanol treatment because both isoproterenol and
forskolin can elevate cAMP, even in the 24-h ethanolexposed cells (Table 1). The function of the cyclases are
not diminished by chronic ethanol, as observed in our
assays. Exposure to ethanol is reversible in washout
studies of ciliary beating. This would also suggest that
100 mM ethanol is not toxic to the cells.
Our control studies indicate that this phenomenon is
not the result of the evaporative loss of ethanol (Fig. 2)
nor is this a function of the metabolic breakdown of
ethanol by alcohol dehydrogenase (ADH). Indeed, we
found little or no presence of ADH activity in the BBEC
(data not shown). We have also found that this desensitization effect occurs with direct or vapor-phase ethanol exposure in vitro (data not shown). Stimulated
increases in enzyme activity are generally accepted to
be significant if the degree of the activity ratio is two or
larger. Indeed, we consistently observed at least a
twofold ethanol-stimulated increase in PKA beginning
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8-BrcAMP, 8-bromo-cAMP; SNP, sodium nitroprusside; 8-BrcGMP, 8-bromo-cGMP.
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CHRONIC ETHANOL DESENSITIZES CILIA
AJP-Lung Cell Mol Physiol • VOL
function of the kinase itself is altered in some way by
long-term ethanol exposure.
There may be several possible explanations for the
modulation of PKA by chronic ethanol. Chronic ethanol
may induce the formation of a PKA inhibitory protein
over time. The rapid nature of desensitization (hours
vs. days) suggests that synthesis of a new PKA inhibitor is unlikely. Chronic ethanol may alter the binding
of cAMP to PKA or may directly affect the active site of
PKA. If cAMP binding is altered because of chronic
ethanol treatment, then 8-BrcAMP would not be able
to activate PKA after the cells have been desensitized
to ethanol. Previous in vitro kinase activity studies
with purified enzyme and substrate suggest that ethanol does not alter the functional structure of the
enzyme (19). Chronic ethanol may interfere with the
autophosphorylation of PKA, thus altering the “priming” of the kinase to acute ethanol. The autophosphorylation of PKA has been shown to increase the enzyme
baseline catalytic activity in response to full activation
by cAMP (33). Chronic ethanol may reduce the autophosphorylation potential of PKA, thus resulting in the
kinase’s inability to bind cAMP in response to an agonist.
In our opinion, the most compelling explanation of
PKA desensitization relates to the compartmentalization of PKA in the BBEC. Chronic ethanol may interfere with the targeting of PKA to its substrate. The
specific compartmentalization of PKA may direct its
ability to phosphorylate substrate resulting in increased CBF. If chronic ethanol blocks or alters the
targeting of PKA to this substrate, it could result in no
PKA activity or CBF increases. Therefore, it may be
useful to localize PKA in the cell under conditions of
ethanol treatment and identify potential cellular substrates for PKA.
In summary, we have found that chronic ethanol
exposure downregulates PKA in airway epithelium.
This is likely an important mechanism by which ethanol blunts the airway’s responsiveness to normal and
pathological stimuli. Ethanol’s effect on signal transduction in the lung may cause the impaired host defenses observed after chronic ethanol ingestion. The
seemingly beneficial effects of acute ethanol exposure
(ⱕ6 h) on ciliary motility we originally observed (31)
appear to contradict what is known about host defenses in chronic alcoholics. Our current findings provide a more compelling explanation of how ethanol
likely impairs rather than improves airway host defenses. In this way, ethanol may be a “two-edged
sword” for the ciliated airway cell, since acute exposure
has a very different impact on ciliary motility than
does chronic exposure.
This work was supported by National Institute of Alcohol Abuse
and Alcoholism Grant 5 RO1 AA-08769–07 to J. H. Sisson and a
Veterans Affairs Merit Review Grant to T. A. Wyatt.
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at ⬃1 h of treatment in all experiments. In Fig. 2, there
is an actual increase in the PKA activity ratio, approaching threefold between 4 and 6 h. As speculated,
this difference is most likely the result of the maximal
ethanol stimulus of 100 mM being replaced every hour,
producing a protocol-specific effect. This effect appears
to manifest itself by sustaining the duration and magnitude of maximal PKA activation but not indefinitely.
This would support our hypothesis that no decrease in
ethanol signal could be occurring because of evaporative loss or cellular metabolism, as PKA activity subsides after 6 h in both cases.
The alteration of protein kinases by ethanol is well
described in other systems (4, 7, 21, 22, 28, 34), further
supporting an important role for kinase control of ciliary motility in the airway. Just as the increase in CBF
appears to be tightly coupled to the activation of PKA
in the BBEC, we also observed that prolonged exposure
to ethanol downregulates PKA activity. Furthermore,
once the BBEC have been exposed to desensitizing
concentrations of ethanol, PKA-stimulated CBF remains unresponsive to activation by cAMP analogs and
isoproterenol. This suggests that ethanol has uncoupled the regulatory pathway by which PKA activation
leads to increases in ciliary motility. Interestingly,
ethanol also desensitizes the cells to CBF increases
stimulated by 8-BrcGMP and SNP (Fig. 4). This observation supports our previous findings that both cAMPand cGMP-dependent pathways are responsible for
stimulated increases in CBF (39) and that ethanol
stimulation of CBF involves a dual activation of PKA
and PKG (32). Certainly, there is an NO component to
CBF as first presented by us (31) and supported by the
findings of others (40). The lack of response of cellular
CBF to cGMP elevation in the chronic ethanol model
may also indicate that cGMP signals via PKA in the
ethanol-stimulated airway epithelial cell. We cannot
rule out that cGMP and cAMP increase CBF by a
compound pathway instead of two independent pathways. The precise mechanism of interaction between
these cyclic nucleotide pathways during acute ethanol
treatment is currently under investigation.
The downregulation of ␤-adrenergic receptors in response to ␤-agonists has been well described (12, 23).
However, it does not appear that ethanol-stimulated
PKA and CBF involve the ␤-adrenergic receptor. We
have found that pretreatment of the BBEC with propranolol does not inhibit ethanol-mediated increases in
PKA or CBF (data not shown). Although receptor desensitization would explain the loss of isoproterenolstimulated PKA activity after chronic ethanol exposure, it does not account for the loss of stimulated PKA
and CBF by cAMP agonist analogs in the chronic
ethanol-treated cell. In preliminary studies, we have
observed a small activation of cAMP-phosphodiesterase in response to ethanol, but this does not explain our
cAMP analog observation because 8-BrcAMP should be
resistant to phosphodiesterase activity. Because PKA
remains unresponsive to exogenous activators after
chronic ethanol exposure, our findings suggest that the
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