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
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 EtOH-stimulated 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.
lung; airway; adenosine 3',5'-cyclic monophosphate; downregulation; -agonist; cyclic nucleotide; cAMP-dependent protein kinase
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 conditions 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 (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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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% collagen-coated 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 µM PKA substrate heptapeptide (LRRASLG), 10 µM cAMP, 0.2 mM IBMX, 20 mM magnesium acetate, and 0.2 mM
[-32P]ATP in a 40 mM Tris · HCl buffer (pH
7.5). Samples (20 µl) were added to 50 µl of the above reaction
mixture and incubated for 15 min at 30°C. Spotting 50 µl 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 µl were added to 50 µl of stock reaction
mixture consisting of 40 mM Tris · HCl (pH 7.4), 20 mM
magnesium acetate, 130 µM kemptide (LRRASLG), and 0.2 mM
[-32P]ATP. Reactions were initiated by the addition of
10 µl 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-µl 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 µM heptapeptide
substrate (RKRSRAE) specific for PKG was substituted for kemptide. PKA
inhibitor peptide (15 µM) 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 · min
1 · 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).
![]() |
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 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
conditions. 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 (6 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 µM isoproterenol or 10 µM 8-bromo-cAMP (8-BrcAMP). BBEC pretreated with medium demonstrated significant elevations in CBF upon treatment with either isoproterenol or 8-BrcAMP
(Fig. 4B). In sharp 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 µM sodium nitroprusside (SNP) or 10 µM
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 or 8-BrcAMP (Fig.
5B). As expected, neither 10 µM SNP nor 10 µM 8-BrcGMP
stimulates PKA activity in BBEC.
|
|
|
|
![]() |
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.
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 ethanol-treated BBEC can continue to proliferate and migrate beyond 24 h to close a wound in the in vitro wound-healing 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 long-term ethanol treatment because both isoproterenol and forskolin can elevate cAMP, even in the 24-h ethanol-exposed 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 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 cAMP- and 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 isoproterenol-stimulated 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 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 (6 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.
![]() |
ACKNOWLEDGEMENTS |
---|
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.
![]() |
FOOTNOTES |
---|
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: twyatt{at}unmc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 July 2000; accepted in final form 3 April 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bradford, MM.
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
2.
Chomet, B,
and
Gach BM.
Lobar pneumonia and alcoholism: an analysis of thirty-seven cases.
Am J Med Sci
253:
300-304,
1967[ISI][Medline].
3.
Cornwell, TL,
Soff GA,
Traynor AE,
and
Lincoln TM.
Regulation of the expression of cyclic GMP-dependent protein kinase by cell density in vascular smooth muscle cells.
J Vasc Res
31:
330-337,
1994[ISI][Medline].
4.
DeVito, WJ,
and
Stone S.
Ethanol inhibits prolactin-induced activation of the JAK/STAT pathway in cultured astrocytes.
J Cell Biochem
74:
278-291,
1999[ISI][Medline].
5.
French, SW,
Miyamoto K,
and
Tsukamoto H.
Ethanol-induced hepatic fibrosis in the rat: role of the amount of dietary fat.
Alcohol Clin Exp Res
10:
13S-19S,
1986[Medline].
6.
Green, GM,
and
Kass EH.
Factors influencing the clearace of bacteria by the lung.
J Clin Invest
43:
769-776,
1964[ISI].
7.
Greenberg, SS,
Jie O,
Zhao X,
Wang JF,
and
Giles TD.
The potential mechanism of induction of inducible nitric oxide synthase mRNA in alveolar macrophages by lipopolysaccharide and its suppression by ethanol, in vivo.
Alcohol Clin Exp Res
22:
260S-265S,
1998[ISI][Medline].
8.
Holford, NH.
Clinical pharmacokinetics of ethanol.
Clin Pharmacokinet
13:
273-292,
1987[ISI][Medline].
9.
Jain, B,
Rubinstein I,
Robbins R,
and
Sisson J.
TNF- and IL-1
upregulate nitric oxide-dependent ciliary motility in bovine airway epithelium.
Am J Physiol Lung Cell Mol Physiol
268:
L911-L917,
1995
10.
Jain, B,
Rubinstein I,
Robbins RA,
Leise KL,
and
Sisson JH.
Modulation of airway epithelial cell ciliary beat frequency by nitric oxide.
Biochem Biophys Res Commun
191:
83-88,
1993[ISI][Medline].
11.
Jiang, H,
Colbran JL,
Francis SH,
and
Corbin JD.
Direct evidence for cross-activation of cGMP-dependent protein kinase by cAMP in pig coronary arteries.
J Biol Chem
267:
1015-1019,
1992
12.
Kelsen, SG,
Higgins NC,
Zhou S,
Mardini IA,
and
Benovic JL.
Expression and function of the beta-adrenergic receptor coupled-adenylyl cyclase system on human airway epithelial cells.
Am J Respir Crit Care Med
152:
1774-1783,
1995[Abstract].
13.
Kim, JS,
McKinnis VS,
Nawrocki A,
and
White SR.
Stimulation of migration and wound repair of guinea-pig airway epithelial cells in response to epidermal growth factor.
Am J Respir Cell Mol Biol
18:
66-74,
1998
14.
Labianca, DA,
and
Simpson G.
Medicolegal alcohol determination: variability of the blood- to breath- alcohol ratio and its effect on reported breath-alcohol concentrations.
Eur J Clin Chem Clin Biochem
33:
919-925,
1995[ISI][Medline].
15.
Lebowitz, MD.
Respiratory symptoms and disease related to alcohol consumption.
Am Rev Respir Dis
123:
16-19,
1981[ISI][Medline].
16.
Lechner, JF,
and
LaVeck MA.
A serum-free method for culturing normal human bronchial epithelial cells at clonal density.
J Tissue Culture Meth
9:
43-48,
1985.
17.
Leitch, GJ,
Frid LH,
and
Phoenix D.
The effects of ethanol on mucociliary clearance.
Alcohol Clin Exp Res
9:
277-280,
1985[ISI][Medline].
18.
Lincoln, TM,
Flockhart DA,
and
Corbin JD.
Studies on the structure and mechanism of activation of the guanosine 3',5'-monophosphate-dependent protein kinase.
J Cell Biol
253:
6002-6009,
1978.
19.
Machu, TK,
Olsen RW,
and
Browning MD.
Ethanol has no effect on cAMP-dependent protein kinase-, protein kinase C-, or Ca(2+)-calmodulin-dependent protein kinase II-stimulated phosphorylation of highly purified substrates in vitro.
Alcohol Clin Exp Res
15:
1040-1044,
1991[ISI][Medline].
20.
Mardini, IA,
Higgins NC,
Zhou S,
Benovic JL,
and
Kelsen SG.
Functional behavior of the beta-adrenergic receptor-adenylyl cyclase system in rabbit airway epithelium.
Am J Respir Cell Mol Biol
11:
287-295,
1994[Abstract].
21.
Moore, MS,
DeZazzo J,
Luk AY,
Tully T,
Singh CM,
and
Heberlein U.
Ethanol intoxication in Drosophila: genetic and pharmacological evidence for regulation by the cAMP signaling pathway.
Cell
93:
997-1007,
1998[ISI][Medline].
22.
Pandey, SC.
Neuronal signaling systems and ethanol dependence.
Mol Neurobiol
17:
1-15,
1998[ISI][Medline].
23.
Penn, RB,
Panettieri RA, Jr,
and
Benovic JL.
Mechanisms of acute desensitization of the beta 2AR-adenylyl cyclase pathway in human airway smooth muscle.
Am J Respir Cell Mol Biol
19:
338-348,
1998
24.
Rannels, SR,
Beasley A,
and
Corbin JD.
Regulatory subunits of bovine heart and rabbit skeletal muscle cAMP-dependent protein kinase isozymes.
Methods Enzymol
99:
55-62,
1983[ISI][Medline].
25.
Roskoski, R.
Assays of protein kinase.
Methods Enzymol
99:
3-6,
1983[ISI][Medline].
26.
Salathe, M,
and
Bookman RJ.
Coupling of [Ca2+]i and ciliary beating in cultured tracheal epithelial cells.
J Cell Sci
108:
431-440,
1995
27.
Sanderson, MJ,
and
Dirksen ER.
Mechanosensitive and beta-adrenergic control of the ciliary beat frequency of mammalian respiratory tract cells in culture.
Am Rev Respir Dis
139:
432-440,
1989[ISI][Medline].
28.
Sapru, MK,
Diamond I,
and
Gordon AS.
Adenosine receptors mediate cellular adaptation to ethanol in NG108-15 cells.
J Pharmacol Exp Ther
271:
542-548,
1994[Abstract].
29.
Shoji, S,
Rickard KA,
Ertl RF,
Linder J,
and
Rennard SI.
Lung fibroblasts produce chemotactic factors for bronchial epithelial cells.
Am J Physiol Lung Cell Mol Physiol
257:
L71-L79,
1989
31.
Sisson, JH.
Ethanol stimulates apparent nitric oxide-dependent ciliary beat frequency in bovine airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol
268:
L596-L600,
1995
32.
Sisson, JH,
May K,
and
Wyatt TA.
Nitric oxide-dependent ethanol stimulation of ciliary motility is linked to cAMP-dependent protein kinase (PKA) activation in bovine bronchial epithelium.
Alcohol Clin Exp Res
23:
1528-1533,
1999[ISI][Medline].
33.
Smith, JA,
Francis SH,
and
Corbin JD.
Autophosphorylation: a salient feature of protein kinases.
Mol Cell Biochem
127/128:
51-70,
1993.
34.
Solem, M,
McMahon T,
and
Messing RO.
Protein kinase A regulates regulates inhibition of N- and P/Q-type calcium channels by ethanol in PC12 cells.
J Pharmacol Exp Ther
282:
1487-1495,
1997
35.
Tamaoki, J,
Tagaya E,
Sakiai N,
and
Kondo A.
Role of nitric oxide in ciliary responses to isoproterenol and bradykinin in rabbit tracheal epithelium (Abstract).
Eur Respir J
7:
12s,
1994.
36.
Wu, R,
Groelke JW,
Chang LY,
Proter ME,
Smith D,
and
Nettesheim P.
Effects of hormones on the multiplication and differentiation of tracheal epithelial cells in culture.
In: Growth of Cells in Hormonally Defined Media, edited by Sirbasku D,
Sato GH,
and Pardee A.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982, p. 641-656.
37.
Wyatt, TA,
Heires AJ,
Sanderson SD,
and
Floreani AA.
Protein kinase C activation is required for cigarette smoke-enhanced C5a-mediated release of interleukin-8 in human bronchial epithelial cells.
Am J Respir Cell Mol Biol
21:
283-288,
1999
38.
Wyatt, TA,
Schmidt SC,
Rennard SI,
and
Sisson JH.
Acetaldehyde-stimulated PKC activity in airway epithelial cells treated with smoke extract from normal and smokeless cigarettes.
Proc Exp Biol Med
225:
91-97,
2000
39.
Wyatt, TA,
Spurzem JR,
May K,
and
Sisson JH.
Regulation of ciliary beat frequency by both PKA and PKG in bovine airway epithelial cells.
Am J Physiol Lung Cell Mol Physiol
275:
L827-L835,
1998
40.
Zhan, X,
Li D,
and
Johns RA.
Immunohistochemical evidence for the NO cGMP signaling pathway in respiratory ciliated epithelia of rat.
J Histochem Cytochem
47:
1369-1374,
1999