 |
INTRODUCTION |
Epidemiological studies suggest that moderate alcohol ingestion
(i.e. 1-2 drinks/day) reduces the risk of
cardiovascular-related disease (1, 2). Although the precise
mechanism(s) by which alcohol protects against atherosclerotic heart
disease is not known, recent studies suggest that some of the
beneficial effects of alcohol may be mediated by its ability to prevent
vascular thrombosis and occlusion (3). Because the rupture of
atherosclerotic plaques with ensuing thrombosis is widely believed to
be the predominant etiology underlying acute coronary syndromes (4),
endogenous mediators such as the proteolytic enzyme, tissue-type
plasminogen activator
(t-PA),1 and its inhibitor,
plasminogen activator inhibitor (PAI)-1 are important in regulating the
thrombotic process (5). The function of t-PA is to convert plasminogen
to a proteolytic enzyme, plasmin, which digests
fibrin-dependent blood clots. Thus, thrombotic vascular complications may be attenuated by agents that increase the secretion of t-PA and/or decrease the expression of PAI-1 (6-8). Indeed, the
plasma level of t-PA has been shown to be inversely correlated with the
risk of myocardial infarction (9).
The induction of t-PA gene transcription is regulated, in part, by cAMP
and transcription factors that bind to activator protein-2 and
shear-stress response element (10-12). The ability of cAMP to mediate
increases in t-PA expression has been demonstrated in many cell types
including hepatocytes, fibroblasts, and endothelial cells (13-16).
Although higher concentrations of alcohol (i.e. >100
mM) have been shown to modulate guanine nucleotide-binding protein (G-protein) activity and cAMP levels in neuronal tissues or
cell lines (17-19), it is not known whether lower concentrations of
alcohol (i.e. <5 mM) have similar effects in
vascular endothelial cells and whether these effects, if present,
translate into specific gene expression that contributes to the
cardiovascular protective effects of moderate alcohol ingestion. Thus,
the purpose of this study is to characterize the effects of alcohol on
G-protein function in vascular endothelial cells and to determine
whether concentrations of alcohol that are associated with the
ingestion of less than two alcoholic beverages can up-regulate t-PA
expression via G-protein-mediated cAMP-dependent signaling pathways.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All standard culture reagents were obtained from
JRH Bioscience. Isoproterenol, propranolol, ascorbic acid, creatinine
phosphate, phosphocreatine kinase, GTP
S, phenylmethylsulfonyl
fluoride, leupeptin, aprotinin, dithiothreitol, bovine serum albumin,
ATP, GDP, GTP, 8-bromo-cAMP, and cholera and pertussis toxins were purchased from Sigma. UK14304 (
2-adrenergic receptor
agonist) was a gift from Pfizer. The radioisotopes,
[
-32P]GTP (30 Ci/mmol), [35S]GTP
S
(1250 Ci/mmol), [
-32P]UTP (800 Ci/mmol), and
[
-32P]CTP (3000 Ci/mmol) and the polyclonal rabbit
antisera to G
s (RM/1) were supplied by NEN Life Science
Products. The polyclonal rabbit antisera P4 was raised against a
purified decapeptide corresponding to the COOH-terminal regions of
G
i2 (20). Protein molecular weight markers were
purchased from Life Technologies, Inc. The chemiluminesence detection
kit (ECL) was obtained from Amersham Pharmacia Biotech. The
polyvinylidene difluoride transfer membrane (pore size 0.2 µm) was
purchased from Bio-Rad. The heterologous 4×-CRE promoter luciferase
reporter construct (p.CRE-Luc) was purchased from Strategene (San
Diego, CA). The full-length human t-PA and PAI-1 cDNAs were
obtained from Sandra J. Degen (University of Cincinnati) and David
Luskatoff (Scripps Institute, La Jolla, CA), respectively.
Cell Culture--
Endothelial cells were harvested from human
saphenous veins and human and bovine aortas as described (21, 22). The
cells were cultured at 37 °C in a growth medium containing
Dulbecco's modified Eagle's medium) supplemented with 5 mM L-glutamine (Life Technologies, Inc.), 10%
fetal calf serum (Hyclone), and an antibiotic mixture of penicillin
(100 units/ml), streptomycin (100 mg/ml), Fungizone (250 ng/ml).
Relatively pure endothelial cell cultures were confirmed by Nomarski
optical microscopy (Olympus IX70, 40 × objective) and by
immunofluorescence staining with anti-Factor VIII antibodies. All
passages were performed with a disposable cell scraper (Costar), and
only endothelial cells of less than six passages were used. Confluent
endothelial cells (~2 × 106 for human and ~5 × 106 for bovine) were treated with various concentrations
of ethanol, and culture dishes were parafilm-wrapped for the indicated
time intervals.
Western Blotting--
Partially purified membranes were prepared
from control and ethanol-treated endothelial cells as described
previously (22). Membrane proteins (25 µg) and molecular weight
markers were separated by SDS/polyacrylamide gel electrophoresis (10%
running, 4% stacking gel). The proteins were electrophoretically
transferred onto polyvinylidene difluoride membranes and incubated
overnight at 4 °C with blocking solution (5% nonfat dry milk and
0.1% Tween 20 in phosphate-buffered saline) before to the addition of
the following dilutions of specific rabbit polyclonal antisera: P4
(1:400) and RM/1 (1:1000). The polyvinylidene difluoride membranes were
then washed twice with phosphate-buffered saline buffer containing
0.1% Tween 20 and then treated with donkey anti-rabbit horseradish
peroxidase antibody (1:4000) (Amersham Pharmacia Biotech). Radiographic
chemiluminescence was performed several times at 23 °C, and the
appropriate exposures were subjected to densitometric analysis.
Northern Blotting--
Equal amounts of total RNA (20 µg) were
separated by 1.2% formaldehyde-agarose gel electrophoresis,
transferred overnight onto Hybond nylon membranes (Amersham Pharmacia
Biotech) by capillary action, and baked for 2 h at 80 °C before
prehybridization. Radiolabeling of full-length human t-PA or PAI-1
cDNA were performed using random hexamer priming,
[
-32P]CTP, and Klenow (Amersham Pharmacia Biotech).
The membranes were hybridized with the indicated probes overnight at
45 °C in a solution containing 50% formamide, 5× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate),
2.5 × Denhardt's solution, 25 mM sodium phosphate
buffer (pH 6.5), 0.1% SDS, and 250 µg/ml salmon sperm DNA. All
Northern blots were subjected to stringent washing conditions (0.2×
SSC, 0.1% SDS at 65 °C) before autoradiography with intensifying
screen at
80 °C for 24-72 h. Equalization of RNA loading was
assessed by re-probing all blots with human
-tubulin (ATCC number 37855).
GTPase Assay--
Membranes (30 µg) from endothelial cells
treated with the indicated conditions were incubated for 90 min at
22 °C in the presence or absence of specific COOH-terminal antisera
before GTPase assay as described (23). Preliminary studies revealed
that maximal inhibition of receptor-G-protein coupling was achieved by
the antisera at the following dilutions: P4 (1:50) and RM/1 (1:100). The GTPase assay was initiated by the addition of isoproterenol (100 nM) or UK14304 (
2-adrenergic receptor
agonist, 10 nM) to the reaction mixture as described
previously (20). Nonspecific activity was determined in the presence of
GTP
S (10 µM) and represented ~10% of total
activity. Isoproterenol- and UK14304-stimulated GTPase activity was
calculated as the difference between total and nonspecific activity and
expressed as pmol/min/mg of membrane protein. Assays were performed in
duplicate with less than 10% variation.
[35S]GTP
S Binding Assay--
Membrane proteins
(30 µg) from control and ethanol-treated endothelial cells were
incubated for 30 min at 30 °C in a buffer containing
[35S]GTP
S (20 nM), GTP (2 µM), MgCl2 (5 mM), EGTA (0.1 mM), NaCl (50 mM), creatine phosphate (4 mM), phosphocreatine kinase (5 units), ATP (0.1 mM), dithiothreitol (1 mM), leupeptin (100 µg/ml), aprotinin (50 µg/ml), bovine serum albumin (0.2%), and
triethanolamine HCl (50 mM, pH 7.4). The assay was
initiated by the addition of isoproterenol (100 nM) or
UK14304 (10 nM) and terminated after 30 min with excess
unlabeled GTP
S (100 µM). Samples were then resuspended
in 100 µl of immunoprecipitation buffer containing Triton X-100
(1%), SDS (0.1%), NaCl (150 mM), EDTA (5 mM),
Tris-HCl (25 mM, pH 7.4), leupeptin (10 µg), aprotinin
(10 µg), and phenylmethanesulfonyl fluoride (2 mM).
The following G-protein antisera with their corresponding final
dilutions were added to the mixture:
i2 (P4, 1:20) and
s (RM/1, 1:100). The samples were allowed to incubate
for 16 h at 4 °C with gentle mixing. The antibody-G-protein
complexes were then incubated with 50 µl of protein A-Sepharose (1 mg/ml, Amersham Pharmacia Biotech) for 2 h at 4 °C, and the
precipitate was collected by centrifugation at 12,000 × g for 10 min. Preliminary studies indicated that all
i2 and
s were completely precipitated by this procedure because Western blot analysis of the supernatant with
the P4 and RM/1 antisera did not reveal the presence of 40-41- or
46-52-kDa proteins, respectively. The pellets were washed three times
in a buffer containing HEPES (50 mM, pH 7.4), NaF (100 µM), sodium phosphate (50 mM), NaCl (100 mM), Triton X-100 (1%), and SDS (0.1%). The final pellet
containing the immunoprecipitated [35S]GTP
S-labeled
G-protein was counted in a liquid scintillation counter (LS 1800, Beckman Instruments, Inc., Fullerton, CA). Nonspecific activity was
determined in the presence of excess unlabeled GTP
S (100 µM).
cAMP Assay--
Confluent endothelial cells were treated for
12 h with the indicated concentrations of ethanol. After the
addition of isobutylmethylxanthine(0.5 mM) for 15 min,
cells were stimulated with isoproterenol (100 nM) in the
presence or absence of propranolol (100 µM) for another 15 min. Unstimulated cells were treated with isobutylmethylxanthine for
30 min. Cells were scraped on ice, pelleted, and resuspended in
ice-cold isobutylmethylxanthine (0.5 mM), boiled for 3 min, and frozen at
70 °C. The intracellular cAMP level was determined using a radioimmunoassay kit with [3H]cAMP (Amersham
Pharmacia Biotech).
Transfection Assays--
Transient transfections in bovine
aortic endothelial cells were accomplished using the calcium-phosphate
precipitation method (21). Preliminary studies with
-galactosidase
staining indicate that the transfection efficiency was ~12%. A
heterologous promoter containing four tandem 4×-CRE elements linked to
the luciferase reporter gene (pCRE-Luc, Stratagene) were used to assess
cAMP-dependent gene transcription. Bovine endothelial cells
(60-70% confluent) were transfected with 5 µg of the pCRE-Luc and 1 µg of pCMV.
-gal construct. Approximately 48 h after
transfection, endothelial cells were treated with ethanol or
isoproterenol, alone or in combination, for 12 h. The luciferase
and
-galactosidase activities were determined by chemiluminescence
(Dual-Light, Tropix, Bedford, MA) using a Berthold L9501 luminometer.
Nuclear Run-on Assay--
Confluent endothelial cells (5 × 107 cells) were treated with ethanol (2.5 mM,
12 h) in the presence or absence of isoproterenol (100 nM). Nuclei were isolated, and in vitro
transcription was performed with [
-32P]UTP (800 Ci/mmol) as described previously (24). Equal amounts (1 µg) of
purified, denatured full-length human t-PA, human,
-tubulin (ATCC n
number 37855), and linearized pGEM-3z cDNA were vacuum-transferred onto nitrocellulose membranes using a slot blot apparatus (Schleicher & Schuell). Hybridization of radiolabeled mRNA transcripts to the
nitrocellulose membranes was carried out at 45 °C for 48 h in a
buffer containing 50% formamide, 5× SSC, 2.5 × Denhardt's solution, 25 mM sodium phosphate buffer (pH 6.5), 0.1%
SDS, and 250 µg/ml salmon sperm DNA. The membranes were then washed
with 1× SSC, 0.1% SDS for 1 h at 65 °C before autoradiography
for 72 h at
80 °C. Band intensities were subjected to
analyses by laser densitometry.
Data Analysis--
Band intensities from Northern, Western, and
nuclear run-on assay blots were analyzed densitometrically by the
National Institutes of Health Image Program (25). All values are
expressed as mean ±S.E. compared with controls and among separate
experiments. Paired and unpaired Student's t tests were
employed to determine the significance of changes in GTPase, GTP
binding, and luciferase activities. A significant difference was taken
for p values less than 0.05.
 |
RESULTS |
Cell Culture--
There were no observable adverse effects of
ethanol, isoproterenol, and 8-bromo-cAMP on cell number, morphology, or
immunofluorescent staining for Factor VIII-related antigens. Cellular
confluence and viability as determined by trypan blue exclusion were
maintained for all treatment conditions described.
Effects of Ethanol on t-PA and PAI-1 mRNA
Expression--
Treatment of human vascular endothelial cells with
ethanol (0-2.5 mM, 24 h) alone did not affect
steady-state t-PA mRNA expression (Fig.
1A). However, at an ethanol
concentration of 25 mM, there was a 36 ± 5%
reduction in t-PA mRNA levels compared with basal levels
(p < 0.05, n = 3). Stimulation of
untreated endothelial cells with isoproterenol (100 nM)
increased t-PA mRNA levels by 48 ± 6% (p < 0.05 compared with base line, n = 3). Endothelial cells
treated with 0.25 and 2.5 mM ethanol showed a 2.8- and
2.3-fold increase in isoproterenol-stimulated t-PA mRNA levels
compared with ethanol alone (p < 0.05 for both,
n = 3). At an ethanol concentration of 25 mM, there was no significant increase in
isoproterenol-stimulated t-PA mRNA expression compared with ethanol
alone (p > 0.05, n = 3). Similar
results were observed with human aortic endothelial cells (data not
shown). These results indicate that low concentrations of ethanol (< 10 mM) augment isoproterenol-stimulated t-PA expression in
vascular endothelial cells.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 1.
Northern analyses (20 µg/lane) showing the effects of
ethanol (0-25 mM) alone or in combination with
isoproterenol (ISO, 100 nM) on endothelial
(A) t-PA and (B) PAI-1 mRNA
levels at 24 h. Each blot was reprobed for -tubulin
mRNA for standardization. Each experiment was performed three times
with similar results.
|
|
Treatment of endothelial cells with increasing concentrations of
ethanol (0-25 mM, 24 h) also did not significantly
affect PAI-1 steady-state mRNA levels (Fig. 1B).
However, stimulation with isoproterenol (100 mM) alone
decreased PAI-1 mRNA levels by 28 ± 5% (p < 0.05, n = 3). Ethanol concentrations of 0.25 and 2.5 mM decreased PAI-1 mRNA levels by 40 ± 5 and
65 ± 6%, respectively (p < 0.05 for both,
n = 3) in the presence of isoproterenol. Interestingly, higher concentrations of ethanol (i.e. 25 mM)
produced no further decrease in PAI-1 mRNA levels in the presence
of isoproterenol compared with that of isoproterenol alone (32 ± 4% versus 28 ± 5%, p > 0.05, n = 3). The effects of ethanol on t-PA expression are
almost completely the opposite effects of ethanol on PAI-1 expression.
These findings suggest that the effects of low concentrations of
ethanol favor fibrinolysis by increasing the ratio of t-PA to PAI-1 levels.
Effects of Ethanol on t-PA Gene Transcription--
Nuclear run-on
assays showed that ethanol (2.5 mM, 12 h) alone did
not affect t-PA gene transcription relative to that of
-tubulin
(Fig. 2). Stimulation of untreated
endothelial cells with isoproterenol (100 nM) increased
t-PA gene transcription by 2.2-fold relative to that of
-tubulin
(p < 0.05, n = 2). In endothelial
cells treated with ethanol (2.5 mM) and stimulated with
isoproterenol (100 nM), t-PA gene transcription was
increased by 3.4-fold compared with untreated control cells
(p < 0.1) and increased by 2.8-fold compared with
ethanol treatment alone (p < 0.05). These findings are
consistent with changes in t-PA mRNA expression and suggest that
low concentrations of alcohol augment isoproterenol-stimulated t-PA
gene transcription but, by itself, have very little, if any,
effects.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 2.
Blots from a representative set of nuclear
run-on assays showing the effects of ethanol (2.5 mM) and
isoproterenol (ISO, 100 nM), alone or in
combination, on t-PA gene transcription at 12 h. Band
intensities of t-PA were normalized to the corresponding band
intensities of -tubulin, and the ratio (relative intensity) was set
to a value of 1.0 for the untreated (control) condition. Nonspecific
binding was determined by hybridization to pGEM vector. Experiments
were performed twice with similar results.
|
|
Preliminary studies using different amounts of radiolabeled RNA
transcripts demonstrate that under our experimental conditions, hybridization was linear and nonsaturable. The density of each t-PA
band was standardized to the density of its corresponding
-tubulin
band (relative intensity). The specificity of each band was determined
by the lack of hybridization to the nonspecific pGEM cDNA vector.
Effect of Ethanol on G-protein Expression--
Because
isoproterenol stimulated an increase in t-PA gene transcription and
mRNA expression is dependent upon coupling to specific G-proteins,
particularly G
s, we investigated whether low
concentrations of alcohol affect G-protein expression. Treatment of
endothelial cells with ethanol (0-50 mM, 12 h) did
not affect the protein levels of G
s or
G
i2 (Fig. 3). Furthermore,
stimulation of endothelial cells with the
2-adrenergic
agonist, isoproterenol (100 nM), or the
2-adrenergic agonist, UK14304 (10 nM), had
no effect on G
s and G
i2 expression,
respectively (data not shown).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 3.
Western blots (30 µg
protein/lane) showing the concentration
dependent effects of ethanol (0-50 mM) on
G s and
G i2 protein levels at 12 h. The blots are representative of three separate
experiments.
|
|
The P4 (G
i2) and RM/1 (G
s) antisera were
relatively specific because recognition of their respective
subunits could be blocked in the presence of excess decapeptides from
which they were derived (20, 22). In addition, we found that the amount of
q/11 and common
subunit as determined by the QL
and SW/1 antisera, respectively, was also unaffected by treatment with ethanol (data not shown).
Effect of Ethanol on Agonist-stimulated GTPase Activity--
The
basal GTPase activity in human endothelial cell membrane was 2.5 ± 0.5 pmol/min/mg. Treatment with ethanol (0-25 mM,
12 h) had no effect on basal GTPase activity (Fig.
4A). Stimulation of untreated
endothelial cells (control) with isoproterenol (100 nm) and UK14304 (10 nM) increased GTPase activity by 2.6- and 5.4-fold,
respectively (6.5 ± 1.1 and 13.4 ± 0.7 pmol/min/mg, p < 0.01 compared with basal levels, n = 4). Compared with isoproterenol-stimulated untreated control cells,
ethanol-treated endothelial cells show a progressive increase in
isoproterenol-stimulated GTPase activity, resulting in a maximal
2.2-fold increase in isoproterenol-stimulated GTPase activity at an
ethanol concentration of 2.5 mM (6.5 ± 1.1 to
14.5 ± 1.2 pmol/min/mg, p < 0.01, n = 4). In contrast, ethanol-treated endothelial cells
showed a maximal 38 ± 5% decrease in UK14303-stimulated GTPase
activity at an ethanol concentration of 2.5 mM (13.4 ± 0.7 to 8.3 ± 1.1 pmol/min/mg, p < 0.05, n = 4). Interestingly, higher ethanol concentrations
(i.e. 25 mM) resulted in no further increase or
decrease in isoproterenol- and UK14304-stimulated GTPase activity,
respectively (11.7 ± 1.8 and 10.0 ± 2.1 pmol/min/mg, respectively, p > 0.05 for both, compared with GTPase
values at ethanol concentration of 2.5 mM). In a
time-dependent manner, ethanol (2.5 mM)
increased isoproterenol-stimulated GTPase activity by 290 ± 22%
and decreased UK14304-stimulated GTPase activity by 42 ± 7%,
with maximal effects of both agonists occurring after 12 h of
ethanol exposure (Fig. 4B).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
Concentration (at 12 h (A) and
time-dependent effects (B) of ethanol (0-25
mM) on basal and agonist-stimulated GTPase activity in
endothelial cells. Membranes from ethanol-treated endothelial cells
were stimulated in the absence (panel A, ) or presence
(panel A, ) of isoproterenol (ISO) (100 nM) or UK14304 (UK) ( , 10 nM).
The asterisk represents a significant difference compared
with untreated cells (control). Panel A, N = 4. C) Isoproterenol (100 nM)- or UK14304 (10 µM)-stimulated GTPase activity in endothelial cell
membranes in the absence (None) or presence of antibodies to
the carboxyl terminus of i2 (P4) or
s (RM/1), alone or in combination. The
asterisk represents a significant difference compared with
no antibody treatment (none) for isoproterenol or UK14304.
|
|
Pretreatment with the COOH-terminal antisera to
s (RM/1)
and
i2 (P4) abolished 91 ± 4% (7.2 ± 0.6 to
2.9 ± 0.3 pmol/min/mg) and 93 ± 5% (13.4 ± 1.1 to
3.3 ± 0.4 pmol/min/mg) of isoproterenol- and UK14304-stimulated
GTPase activity, respectively (p > 0.05 for both,
compared with basal unstimulated GTPase activity of 2.5 ± 0.5 pmol/min/mg) (Fig. 4C). In contrast, pretreatment with the
P4 antisera abolished < 10% of isoproterenol-stimulated GTPase activity, whereas the RM/1 antisera decreased <5% of
UK14304-stimulated GTPase activity. Furthermore, isoproterenol- and
UK14304-stimulated GTPase activity were completely blocked with
propranolol (0.1 mM) and rauwolscine (1 µM),
respectively (data not shown).
Effects of Ethanol on Agonst-stimulated GTP
S-Binding
Activity--
To determine the effects of ethanol on specific
G-protein
subunit GTP-binding activity,
s and
i2 from ethanol-treated membranes were radiolabeled with
[35S]GTP
S and immunoprecipitated with specific
antisera directed against
s and
i2
subunits. Treatment with ethanol (0-25 mM, 12 h)
alone did not significantly affect basal
s and
i2 GTP binding activity (p > 0.05 for
both). Endothelial cells stimulated with isoproterenol (100 nM) and UK14304 (10 nM) have
s
and
i2 GTP binding activity of 2.0 ± 0.3 and
17.1 ± 1.3 fmol/min/mg, respectively (Fig.
5). Treatment with ethanol (0-25
mM, 12 h) caused a progressive increase in
s GTP binding activity with a maximal 2.9-fold increase
(5.9 ± 0.7 fmol/min/mg, p < 0.05, n = 3) occurring at an ethanol concentration of 2.5 mM. In contrast, the same membrane from ethanol-treated
cells exhibited a progressive decrease in
i2 GTP binding
activity with a maximal 80 ± 5% decrease (3.5 ± 0.3 fmol/min/mg, p < 0.01, n = 3), also
occurring at an ethanol concentration of 2.5 mM.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 5.
Specific G-protein activity as determined by
immunoprecipitation of isoproterenol (100 nM)-stimulated
[35S]GTP S-labeling of
G s and UK14304 (10 nM)-stimulated
[35S]GTP S-labeling of
G i2 at 12 h. The
asterisk represents a significant difference compared with
untreated endothelial cells (no ethanol).
|
|
Effects of Ethanol on Adenylyl Cyclase Activity--
To determine
whether the effects of ethanol on G
s and
G
i2 correlate with downstream effector activity
(i.e. adenylyl cyclase), we measured intracellular cAMP
levels in ethanol-treated endothelial cells. Treatment with ethanol
(0-25 mM, 12 h) did not affect basal cAMP levels
(Fig. 5). Stimulation with isoproterenol (100 nM) increased
cAMP level from 2.7 ± 0.2 to 4.9 ± 0.3 pmol/500,000 cells
(p < 0.05, n = 4) (Fig.
6). This increase in
isoproterenol-stimulated cAMP level was completely blocked by
propranolol (2.9 ± 0.4 pmol/500,000 cells, p > 0.05 compared with basal levels).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 6.
Concentration-dependent effects
of ethanol (0-25 mM, 12 h) on intracellular cAMP
levels in response to no stimulation (None),
isoproterenol (ISO, 100 nM), or
isoproterenol (100 nM) and propranolol
(Prop, 100 µM). The asterisk
represents a significant difference compared with no stimulation.
Double asterisks represent a significant difference compared with
isoproterenol-stimulated untreated endothelial cells (no
ethanol).
|
|
Endothelial cells treated with ethanol (2.5 mM, 12 h)
showed a significant increase in isoproterenol-stimulated cAMP level (2.7 ± 0.2 to 8.2 ± 0.5 pmol/500,000 cells,
p < 0.05). This increase represented a 2.5-fold
increase in absolute isoproterenol-stimulated cAMP level (2.2 ± 0.2 to 5.5 ± 0.3 pmol/500,000 cells, p < 0.05). Interestingly, lower concentrations of ethanol (0.25 mM,
12 h) did not produce a significant increase in
isoproterenol-stimulated cAMP level compared with untreated cells, and
higher concentrations of ethanol (25 mM, 12 h) did not
produce further increases in isoproterenol-stimulated cAMP level
compared with cells treated with 2.5 mM ethanol. These
findings are consistent with the effects of alcohol on t-PA expression
and suggest that low concentrations of alcohol augment
isoproterenol-stimulated cAMP-dependent t-PA gene transcription.
Effects of Ethanol on cAMP-dependent Promoter
Activity--
The cAMP-dependent gene transcription
involves transcription factors that bind to the cAMP response element
(CRE) of target genes (26). To determine whether increases in cAMP
levels produced by low concentrations of ethanol are sufficient to
transactivate promoters containing CRE, we transfected endothelial
cells with a heterologous luciferase reporter construct containing four
tandem CREs (p.CRE-Luc). Treatment of transfected endothelial cells
with isoproterenol (100 nM, 12 h) or 8-bromo-cAMP (10 µM, 12 h) increased p.CRE-Luc activity by 3.7- and
3.3-fold, respectively (p < 0.05 for both,
n = 3) (data not shown).
Treatment of transfected endothelial cells with ethanol (0-50
mM, 12 h) alone did not affect basal p.CRE-Luc
activity. However, stimulation of ethanol-treated transfected cells
with isoproterenol (100 nm) augmented p.CRE-Luc activity compared with
that of isoproterenol alone (Fig.
7A). The maximal
isoproterenol-stimulated p.CRE-Luc activity in ethanol-treated cells
was 6.9- and 2.1-fold higher than that of untreated and
isoproterenol-stimulated control cells, respectively, and occurred at
an ethanol concentration of 2.5 mM. Higher concentrations
of ethanol (25-50 mM, 12 h) resulted in reductions
in isoproterenol-stimulated p.CRE-Luc activity. In a
time-dependent manner, ethanol (2.5 mM)
augmented isoproterenol-stimulated p.CRE-Luc activity compared with
that of untreated endothelial cells (5.6-fold versus
3.5-fold, respectively, p < 0.05) (Fig. 7B). The increase in isoproterenol-stimulated p.CRE-Luc
activity occurred after 12 h of ethanol exposure, and this time
point is consistent with the maximal increase in
isoproterenol-stimulated G
s GTPase activity.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Concentration (at 12 h (A) and
time-dependent effects (B) of ethanol (0-50
mM, 0-24 h) on basal (None) and isoproterenol
(ISO, 100 nM)-stimulated CRE promoter activity
(fold induction). For the time course, isoproterenol-stimulated CRE
promoter activity was performed in untreated (control) or ethanol (2.5 mM)-treated endothelial cells for the indicated time
points. Transfection efficiency was standardized to -galactosidase
activity, and changes in the ratio of CRE promoter activity to
-galactosidase activity were calculated relative to basal activity
(fold induction). The asterisk represents a significant
difference compared with untreated cells.
|
|
 |
DISCUSSION |
We have shown that alcohol concentrations corresponding to
moderate ingestion (i.e <10 mM) augment agonist-stimulated
endothelial t-PA gene transcription via G-protein-mediated
cAMPdependent pathway. Furthermore, these effects of alcohol
were associated with a reciprocal decrease in agonist-stimulated PAI-1
expression. Higher concentrations of alcohol (
25 mM),
however, did not produce further increases in intracellular cAMP
levels, but instead, decreased isoproterenol-stimulated GTPase activity
and t-PA expression and increased PAI-1 expression. Interestingly,
treatment with alcohol alone did not affect basal G-protein activity,
intracellular cAMP level, p.CRE-Luc activity, or t-PA gene
transcription and expression. These findings are in contrast to
previous studies showing that alcohol decreased G
s
expression and adenylyl cyclase activity in brain tissues (17, 27).
However, in these previous studies, higher concentrations of alcohol
were used for a much longer duration. Our results, therefore, indicate
that alcohol at concentrations associated with moderate ingestion
augments
2-adrenergic receptor-stimulated intracellular
cAMP level and t-PA gene transcription.
The effect of alcohol on isoproterenol-stimulated G
s
activity is rather specific because alcohol concomitantly
down-regulated UK14304-stimulated GTPase activity. Because the
2-adrenergic receptor agonist, UK14304, is predominantly
coupled to G
i2 in vascular endothelial cells (28), these
results suggest that alcohol differentially modulates G
s
and G
i2 activity. Because there were no observable
changes in the amounts of G
s and G
i2, our
results also indicate that alcohol modulates G-protein function rather
than expression. Specificity in receptor-G-protein coupling was
confirmed by [35S]GTP
S binding of specific G-protein
subunits and with studies using carboxyl-terminal-directed
antibodies to G
s and G
i2 that specifically blocked isoproterenol- and UK14304-stimulated GTPase activity, respectively. Thus, the net effects of alcohol on
G
s and G
i2 activity is to favor
receptor-mediated activation of adenylyl cyclase. However, there was no
direct stimulatory effect of alcohol on adenylyl cyclase activity,
because alcohol alone was unable to increase intracellular cAMP level.
The regulation of gene transcription by cAMP is mediated by
trans-acting factors, which bind to CRE (5'-ATGACGTCAT-3')
of target genes (26). Although the consensus sequence for CRE is not
present in the t-PA promoter, a functional CRE-like element has been
identified that acts synergistically with a putative activator
protein-2 binding site to induced t-PA gene transcription by phorbol
12-myristate 13-acetate (29). Indeed, previous studies have shown that
cholera toxin and dibutyryl-cAMP can directly induce t-PA gene
transcription in rat hepatocytes (13), although in HeLa cells, the
activation of cAMP-dependent transcription factor(s) alone
is not sufficient to transactivate the t-PA promoter (14, 29). These
studies, therefore, suggest that other cis-acting element(s)
may play an important role in transactivating the t-PA promoter.
Interestingly, a recent study by Grenett et al. (30) showed
that alcohol alone can increase t-PA gene transcription. Although we
did not observe an increase in t-PA gene transcription and expression
with alcohol alone, there are subtle differences between the two
studies. In contrast to the previous study, our study utilized lower
concentrations of alcohol (i.e. 2.5 mM) for a
longer duration (i.e. up to 24 h). Furthermore, our
finding that alcohol alone does not increase t-PA gene transcription is consistent with the other findings of this study showing that alcohol
alone does not stimulate G
s activity, cAMP production, or CRE promoter activity.
The precise mechanism by which alcohol alters membrane signal
transduction and increases t-PA expression is not known. Previous studies have shown that high concentrations of alcohol (i.e.
100 mM) increase
2-adrenergic and muscarinic
receptor expression in a neuronal NG108-15 cells (31). Although we
cannot exclude the possibility that alcohol may have induced similar
changes in
2-adrenergic receptor affinity and density in
our study, these effects of alcohol, however, are unlikely given the
relatively low concentrations of alcohol used and the lack of
significant changes in the EC50 (~1 nM) of
isoproterenol with respect to pCRE-Luc activity in alcohol-treated
cells. It is also possible that with longer duration, alcohol, even at
low concentrations, may ultimately induce changes in the level of
G
s and G
i2. Indeed, chronic alcohol treatment, albeit at high concentrations, have been shown to inhibit adenylyl cyclase activity via increasing G
i and
decreasing G
s expression in neuronal cells (17, 32).
Under the conditions of our study, however, the observed changes in
isoproterenol-stimulated G-protein activity, cAMP levels, and t-PA gene
expression occurred in the absence of significant changes in
endothelial G
s and G
i2 expression.
The etiology of acute coronary syndromes is thought to be because of
plaque rupture with ensuing vascular thrombosis and occlusion (4). The
degree of vascular thrombosis and thrombolysis is regulated, in part,
by the level of t-PA and PAI-1. Recent epidemiology studies suggest
that moderate alcohol consumption (i.e. 1-2 drinks/day) is
associated with higher serum t-PA levels and lower incidence of
myocardial infarctions (1, 2, 9). Because the ingestion of two glasses
of wine with an alcoholic content of 4% represents an alcohol
concentration of approximately 7 mM in vivo
(33), our findings suggest that alcohol may lower the risk of
cardiovascular disease by augmenting t-PA level and inhibiting PAI-1
expression. Both of these effects of alcohol would enhance the
fibrinolytic activity in the vessel wall.
In summary, we have provided a potential mechanism by which low
concentrations of alcohol may protect against atherosclerotic heart
disease. Because alcohol alone does not induce, but rather, augments
agonist-stimulated t-PA gene transcription via
G
s-mediated cAMP-dependent pathway, we
speculate that moderate alcohol consumption may be more beneficial in
terms of t-PA production in active individuals with frequent elevations
in catecholamine levels. Consequently,
-blocker therapy, which is
beneficial in cardiac ischemia, may paradoxically diminish some of the
antithrombotic effects of alcohol on the vessel wall. We propose that
low but not high concentrations of alcohol up-regulate
agonist-stimulated cAMP and t-PA expression through differential
effects on G
s and G
i2 activity. It
remains to be determined, however, the mechanism by which alcohol
modulates the activities of specific G proteins.