Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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Ethanol impairs
hormone-stimulated cAMP production in a number of cell types, yet the
effects of ethanol on downstream responses mediated by cAMP-dependent
protein kinase (PKA) are not understood. Here we have investigated the
effects of ethanol feeding on cAMP-mediated inhibition of tumor
necrosis factor- (TNF-
) synthesis in rat Kupffer cells. Male
Wistar rats were fed liquid diets containing 36% of calories as
ethanol for 4 wk or were pair fed a control diet. Stimulation of cAMP
production by the adenosine A2
receptor agonist 5'-(N-ethylcarboxamido)-adenosine (NECA),
prostaglandin E2, or forskolin was
decreased to 25% of control values in Kupffer cells isolated from
ethanol-fed rats. This decrease was associated with a reduction in the
quantity of immunoreactive Gs
protein in ethanol-fed rats, with no changes observed in
Gi
or G
. TNF-
production
was higher in ethanol-fed rats in response to stimulation with
lipopolysaccharide or latex beads. Despite the profound reduction in
the ability of hormone to increase cAMP production, NECA and prostaglandin E2 inhibited TNF-
production to an equivalent degree in Kupffer cells from ethanol- and
pair-fed rats. Total activity and immuoreactive protein quantity of PKA
did not differ between groups. Activation of PKA in response to a
15-min treatment with 1 µM NECA was reduced by 50% in ethanol-fed
rats compared with control. Despite this reduction in activation,
translocation of the catalytic subunit of PKA to the nucleus and
phosphorylation of cAMP response element binding protein in response to
activation were observed in Kupffer cells from both ethanol- and
pair-fed rats. These data demonstrate that there is a dissociation
between ethanol-induced desensitization of hormone-stimulated cAMP
production in rat Kupffer cells and the downstream inhibition of
TNF-
production mediated by cAMP.
G protein; tumor necrosis factor- ; desensitization; lipopolysaccharide; macrophage; adenosine 3',5'-cyclic
monophosphate
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INTRODUCTION |
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cAMP IS AN important regulator of cellular metabolism, gene transcription, differentiation, and proliferation. Ethanol disrupts hormone-stimulated cAMP production in almost all cell types examined. In many cells types, long-term exposure to ethanol both in vivo and in culture decreases cAMP production via receptors coupled to the stimulatory G protein, Gs, producing a heterologous desensitization (7). However, the interaction of ethanol with cAMP signalling pathways can vary among cell types. For example, long-term treatment of primary cultures of hepatocytes with ethanol increases receptor-stimulated cAMP production (29, 30). Similarly, long-term ethanol feeding in vivo increases cAMP production in liver membranes (3, 20) and adipocytes (41). Despite these clear effects of ethanol on cAMP production, very little is known about the consequences of these changes in cAMP production on downstream responses mediated by cAMP-dependent protein kinase (PKA). cAMP binds to the regulatory subunits (R) of PKA, inducing holoenzyme dissociation and the liberation of the catalytic subunit (PKA-C). Nuclear translocation of PKA-C is the rate-limiting step in the coupling of hormonal stimulation and cAMP-dependent gene transcription (16). Although activity of purified PKA is not affected by ethanol in vitro (24), chronic ethanol exposure decreases PKA activity in the embryonic chick brain (2) and rat striatum (34). Recent data also indicate that phosphorylation of the nuclear transcription factor cAMP response element binding protein (CREB) is increased after short-term, but not long-term, ethanol exposure in rat cerebellum (43). In contrast, treatment of NG108-15 neuroblastoma X glioma cell hybrids with ethanol results in the translocation of the C subunit to the nucleus, independent of hormonal activation (8). However, it is not known whether the C subunit is active under these conditions.
To investigate the relationship between ethanol-induced desensitization
of cAMP production and downstream responses mediated by PKA, we have
utilized a primary cell culture model of Kupffer cells. Kupffer cells,
the resident macrophages in the liver, release fibrogenic and
inflammatory cytokines, as well as superoxide anions, in response to
bacterial lipopolysaccharide (LPS) or phagocytosis of cellular debris
(42). Tumor necrosis factor- (TNF-
) is a major cytokine produced
by Kupffer cells after exposure to LPS (33). cAMP mediates a feedback
inhibitory pathway that decreases TNF-
production in Kupffer cells
(33); elevation of cAMP in Kupffer cells by treatment with
PGE2,
-adrenergic agonists,
adenosine A2 receptor agonists,
and phosphodiesterase inhibitors decreases TNF-
and
interleukin-1 production (4, 6, 13, 32, 35).
Activation of Kupffer cells is likely to play an important role in the
development of localized inflammation and fibrosis in alcoholic liver
disease, since inactivation of Kupffer cells by treatment with
gadolinium chloride can prevent alcohol-induced liver injury in rats
(1). Despite their recognized importance in the etiology of disease,
very little is known about the effects of ethanol on Kupffer cell
function. Therefore, we have investigated the effects of ethanol on
hormone-stimulated cAMP production and subsequent inhibition of TNF-
production in Kupffer cells. By measuring the ability of hormones to
inhibit TNF-
production, we were able to assess the impact
of ethanol-induced desensitization of cAMP production on regulation of
downstream responses mediated by PKA.
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MATERIALS AND METHODS |
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Materials. Adult male Wistar rats
weighing 150 g were purchased from Harlan Sprague-Dawley (Indianapolis,
IN). Liquid diet ingredients were as described (20).
125I-labeled cAMP was from ICN
(St. Laurent, Quebec, Canada). Adenosine receptor agonists were
purchased from Research Biochemicals (Natick, MA).
[32P]ATP and the
enhanced chemiluminescence detection kit were from Amersham (Oakville,
Ontario, Canada). Antibodies to cAMP,
Gs, Gi
, and the
-subunit were
from Gramsch Laboratories (Felsenfeldbruch, Germany) and New England
Nuclear DuPont (Mississauga, Ontario, Canada). Adenosine deaminase,
creatine kinase, human recombinant TNF-
, and the anti-mouse and
anti-rabbit IgG-peroxidase antibodies were from Boehringer
Mannheim (Dorval, Quebec, Canada). The PKA assay system was from GIBCO
BRL (Burlington, Ontario, Canada). Monoclonal antibody to type I
regulatory subunit of PKA (RI) was from Transduction Laboratories
(Lexington, KY), and polyclonal antibodies to type II regulatory
subunit of PKA (RII), phospho-CREB, and PKA-C were from Upstate
Biotechnology (Lake Placid, NY). Ro 20-1724 was from
Biomol Research Laboratories (Plymouth Meeting, PA). Texas
red-conjugated anti-rabbit IgG and sytox green (nuclear stain) were
from Molecular Probes (Eugene, OR). Antibodies to ED1 and ED2 were from
Serotec (Toronto, Ontario, Canada). Other reagent grade
chemicals were from Fisher Scientific (Missisaugua, Ontario, Canada) or
Sigma (St. Louis, MO).
Animals and diets. Procedures using animals were approved by the Animal Care Committee at the University of Guelph or Case Western Reserve University. Adult male Wistar rats were housed in a temperature- and humidity-controlled environment with alternating 12:12-h light-dark cycles. After a 1-wk acclimatization period on standard rat chow, the animals were allowed free access to the LieberDeCarli liquid diet (23) containing 36% of calories from ethanol or were pair fed an identical isocaloric diet in which maltose dextrins were substituted for ethanol. After 4 wk, the animals were anesthetized with pentobarbital sodium, and Kupffer cells were isolated.
Livers were perfused via the portal vein first with modified Hanks'
solution (free of Ca2+ and
Mg2+) containing 1 mM EGTA and
10 mM HEPES and then with 0.05% collagenase (type I) in Williams E
medium with 10 mM HEPES at a flow rate of 15 ml/min. A cell suspension
was formed by gentle disruption of the collagenase-treated livers. The
resulting suspension of cells was then treated further with 0.02%
Pronase and 0.001% DNase for 15 min at 10°C (12). The cell
suspension was then filtered over nylon (230- and 120-µm mesh) and
washed two times by centrifugation at 500 g for 7 min in DMEM with 7.5% fetal
bovine serum and 0.001% DNase. The final cell pellet was resuspended
in Gey's balanced salt solution (GBSS) and 0.001% DNase, layered on a
gradient containing 17.5 and 8.5% nycodenz, and centrifuged for 17 min
at 1,500 g. In some control
experiments, the collagenase-treated liver was not treated with
Pronase. Instead, the liver cell suspension was centrifuged three times
at 50 g for 5 min to pellet
hepatocytes, and the supernatant was collected after each
centrifugation. The pooled supernatant was then centrifuged at 500 g for 10 min to collect nonparenchymal
cells. Kupffer cells were separated from this pellet on nycodenz as
described above. The cells sedimenting between these two densities were
collected by centrifugation at 500 g
for 7 min and resuspended in 10 ml GBSS with 0.001% DNase. This sample
was loaded on a centrifugal elutriator (2,500 rpm). Kupffer cells were
collected at a flow rate of 45 ml/min. Isolated Kupffer cells were
washed two times by centrifugation at 500 g for 7 min. Cells were either used
immediately, plated for cell culture, or frozen at 70°C for
future analysis. Cell viability was measured by trypan blue exclusion,
and Kupffer cells were identified by endogenous peroxidase or naphthyl
esterase activity (28) or the presence of the antigenic macrophage
markers ED1 and ED2.
Cell culture. Isolated Kupffer cells
were suspended in Iscove's modified Dulbecco's medium (IMDM) with
20% fetal bovine serum and penicillin-streptomycin at a concentration
of 2 × 106 cells/ml and
plated on tissue culture plates (96 well for TNF- assay, 12 well for
cAMP and PKA measurements, and LabTek chamber slides for
immunohistochemistry). After 2 h, nonadherent cells were removed by
aspiration, and fresh medium was supplied. Assays were carried out
after 24 h in culture. Attached Kupffer cell number was constant over
the 24-h culture period (data not shown).
Measurement of cAMP production. cAMP
production in intact Kupffer cells was measured after 24 h in culture.
Culture medium was aspirated, and cells were incubated in PBS with 25 mM HEPES, 10 mM glucose, 0.7 mM
CaCl2, and 0.4 mM
MgCl2, pH 7.4 (PBS), containing 1 µM Ro 20-1724, an inhibitor of phosphodiesterase, and 1 U/ml adenosine deaminase to remove any endogenously produced adenosine. Cells were then treated with and without agonists for 10 min as described in Figs. 1-6. Reactions were terminated by the addition of 0.1% Nonidet P-40 and 0.1 N HCl. Cells were incubated on ice for 10 min, and supernatants were removed and stored at 20°C for
subsequent analysis for cAMP by RIA.
Western blot analysis. Kupffer cell
homogenates or crude membrane preparations (29) were diluted to 1 mg
protein/ml in SDS sample buffer. For the detection of
Gi,
Gs
, and G
, 50 µg of crude
membrane proteins were separated by SDS-PAGE using the Laemmli buffer
system (22). Detection of PKA subunits was carried out in homogenates
of cultured Kupffer cells. Proteins were then transferred to
polyvinylidene difluoride membranes, and Western blotting was carried
out as previously described (37). Immunoreactive protein quantity was
assessed by scanning densitometry.
TNF- assay. TNF-
secreted in the Kupffer cell culture medium was measured using a
cytotoxicity assay. After culture for 20 h, cells were treated with LPS
(Escherichia coli 026:B6)
and/or receptor agonists as described in the Figs. 1-6.
After a further 4 h in culture, cell culture medium was removed and
stored at
20°C. Serial dilutions of the conditioned cell
culture medium were added at 100-µl volumes to 96-well culture
dishes. WEHI 164 cells (100 µl), which are sensitive to TNF-
cytotoxicity, were added to each well at 5 × 105 cells/ml in IMDM containing
10% FBS and 0.5 µg/ml actinomycin D. Standard curves were generated
in each plate using human recombinant TNF-
. The plates were cultured
for 20 h, and then 20 µl of
3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium
bromide were added to each well. After 4 h, 150 µl of medium were
removed from each well, and 100 µl of isopropanol-0.04 N HCl were
added to develop metabolized crystals. The plates were then stored in
the dark at room temperature overnight, and absorbance of each well at
550 nm was read in an ELISA plate reader. The concentration of TNF-
in the Kupffer cell medium was calculated based on the standard curve
and was corrected for cell concentration in the original Kupffer cell culture.
Determination of PKA activity.
Activity of PKA, as well as stimulation in response to
106 M
5'-(N-ethylcarboxamido)-adenosine (NECA), was measured by
incubating cells with and without
10
6 NECA for 15 min in PBS.
Reactions were terminated by the addition of ice-cold 10 mM potassium
phosphate, pH 6.8, 10 mM EDTA, 0.5 mM isobutyl methylxanthine, 150 mM
KCl, and 1% Triton X-100. Cells were extracted on ice for 30 min, and
activity of PKA was assayed immediately. PKA activity was measured
using the kemptide assay over a 5-min period in the presence (for total
activity) or absence (for basal or hormone-activated activity) of 40 µM dibutyryl-cAMP. Nonspecific activity was measured by the addition
of 4 µM protein kinase inhibitor peptide.
Immunohistochemistry. Kupffer cells
cultured on LabTek chamber slides were washed two times in PBS and
stimulated with or without
106 M NECA or 10 nM
forskolin for 30 min. After stimulation, cells were fixed for 30 min in
4% freshly prepared paraformaldehyde. Some cells were preincubated
with the fluorescent nuclear stain sytox green for 30 min before
fixation to visualize the nucleus. Slides were then quenched by two
1-min washes in 25 mM glycine and one 5-min wash in PBS and were
blocked in PBS with 2% BSA, 5% fish gelatin, and 0.02% saponin for 2 h at room temperature. Slides were then incubated with antibody to the
PKA-C (1:200) or phospho-CREB (1:200) in blocking buffer overnight at
4°C. Slides were washed three times for 15 min in fresh blocking
buffer and then incubated for 1 h with Texas red-conjugated anti-rabbit
IgG (1:200) for 1 h in blocking buffer. Slides were finally washed three times for 15 min in blocking buffer and two times for 5 min in
PBS. Slides were then mounted and examined by laser scanning confocal
microscopy under a ×60 lens. Nonspecific binding was determined
by incubation with secondary antibody alone.
Statistical analysis. Because of the limited number of Kupffer cells available from each animal, data from several feeding trials are presented in this study; each trial consisted of five rats per feeding group. Values reported are means ± SE. Data were analyzed by Student's t-test or the general linear models procedure blocking for trial effects if data from more than one trial were utilized. Differences between diet groups were assessed by Tukey's test or least-square means. Statistical analyses were performed on the SAS statistical package for the personal computer.
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RESULTS |
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Kupffer cell isolation procedure. To
understand the effects of ethanol on receptor-stimulated cAMP
production in Kupffer cells, we first optimized the Kupffer cell
isolation procedures to ensure that receptor-mediated cAMP production
was not impaired. The standard procedure for Kupffer cell isolation
involves digestion with Pronase, a relatively nonselective protease, to
destroy hepatocytes contaminating the preparation. Because
hormone/cytokine receptors are exposed at the cell surface, Pronase
digestion may impair their function. Therefore, we measured
receptor-stimulated cAMP production in Kupffer cells isolated under
differing Pronase concentrations for increasing periods of incubation
compared with Kupffer cells isolated in the absence of Pronase
(collagenase only; Table 1). At low Pronase
concentrations, short digestion times, and lower digestion temperatures
(data not shown), receptor-stimulated cAMP signal transduction and
plating efficiency in Pronase-treated Kupffer cells were similar to
Kupffer cells isolated without Pronase, with a fivefold increase in
Kupffer cell yield (Table 1). cAMP production after 24 h in culture
increased relative to freshly isolated cells and did not differ between
cells isolated in the absence of Pronase and 0.02% 15-min Pronase
exposure (data not shown).
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Receptor-stimulated cAMP production in Kupffer
cells. Rats were allowed free access to the
Lieber-DeCarli liquid diet containing 36% of calories from ethanol or
were pair fed a control diet that isocalorically substituted maltose
dextrins for ethanol. Growth of the rats on these diets was equivalent
(Table 2). Kupffer cell yields, viability,
plating efficiency, and cell numbers after 24 h in culture were also
equivalent between the two treatment groups (Table 2).
Gs and
Gi
protein quantity was
measured by Western blotting in freshly isolated Kupffer cells from
ethanol and pair-fed rats. Kupffer cells from both feeding groups
expressed the long and short forms of
Gs
, but the total quantity of
Gs
immunoreactivity (long and
short forms combined) was 41% less than in ethanol-fed rats compared
with pair-fed rats (Table 3). The quantity
of Gi
was not affected by
ethanol feeding. The decrease in quantity of
Gs
in Kupffer cells from
ethanol-fed rats was maintained during 24 h of culture in the absence
of ethanol (Fig. 1 and Table 3). After
culture for 24 h, the quantity of immunoreactive Gi
and G
did not differ
between the groups (Table 3). To test the functional significance of
this decrease in Gs
,
receptor-stimulated cAMP production was measured in intact Kupffer
cells. Basal cAMP production did not differ between groups (Fig.
2). However, stimulation of cAMP production
by agents that activate adenylyl cyclase independently of receptors
(forskolin), as well as via adenosine
A2 receptor and
PGE2 receptor (Fig. 2), was lower
in Kupffer cells from ethanol-fed rats. These data indicate that
chronic ethanol feeding decreases receptor-stimulated cAMP production
and that this heterologous desensitization can be observed in Kupffer
cells for at least 24 h in culture.
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Regulation of TNF-
production. The concentration of TNF-
was increased
in Kupffer cells from both ethanol- and pair-fed rats by treatment with
LPS or latex beads for 4 h (Fig.
3A).
TNF-
quantity was greater in nonstimulated (basal) Kupffer cells and in cells exposed to latex beads from ethanol-fed rats compared with
control (Fig. 3A). Similarly, at 400 ng/ml LPS, TNF-
release was greater in ethanol-fed compared with
pair-fed rats, but, after treatment with 4,000 ng/ml LPS, there was no
difference between groups (Fig. 3A).
A second dose response was conducted to evaluate responses to lower LPS
concentrations; TNF-
release was greater in Kupffer cells from
ethanol-fed rats after treatment with 0.4-40 ng/ml LPS compared
with pair-fed rats (Fig. 3B).
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Because the production of cAMP in response to a broad range of
concentrations of both NECA and
PGE2 was reduced in ethanol-fed rats (Fig. 2), we asked whether these agents could modulate TNF- production in cells stimulated with LPS or latex beads. Both
10
4 M NECA and
10
6 M
PGE2 decreased the production of
TNF-
in Kupffer cells from pair-fed rats (Fig.
4). Surprisingly, in ethanol-fed rats, a
similar degree of inhibition was observed when cells were treated with 10
4 M NECA and
10
6 M
PGE2, as well as 1 mM
dibutyryl-cAMP (Fig. 4). Inhibition of TNF-
production was also
equivalent between pair-fed and ethanol-fed animals when Kupffer cells
were pretreated with 10
6 M
NECA. TNF-
production in response to 400 ng/ml LPS was reduced to 63 ± 28% of control in pair-fed rats and 64 ± 14% of control in
ethanol-fed rats (n = 3-4).
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The measurements of cAMP production reported in Fig. 2 were conducted
under conditions designed to assess cAMP synthesis; assays were carried
out over short time periods in the presence of an inhibitor of type IV
phosphodiesterase (Ro 20-1724), the predominant phosphodiesterase
isoform in monocytes (36). In contrast, inhibition of TNF-
production by hormone was measured over longer periods of hormone
treatment and in the absence of phosphodiesterase inhibitor. If ethanol
feeding decreased the activity of phosphodiesterase, cAMP might
accumulate over longer time periods in Kupffer cells from ethanol-fed
rats despite decreased rates of cAMP synthesis. cAMP content was
therefore measured in cultured Kupffer cells after longer periods of
treatment with hormone in the absence of Ro 20-1724. cAMP content was
0.33 ± 0.14 and 0.07 ± 0.01 pmol/106 cells after 15 min and
1.1 ± 0.35 and 0.09 ± 0.05 after 60 min of stimulation with
10
6 M NECA in pair-fed and
ethanol-fed rats, respectively (n = 3-4, P < 0.05 for ethanol-fed
compared with pair-fed rats), indicating that the cAMP content of
Kupffer cells from ethanol-fed rats reflected the decreased rate of
cAMP synthesis.
We also investigated the effects of inhibition of phosphodiesterase
activity on TNF- production in Kupffer cells from ethanol- and
pair-fed rats. If a decrease in phosphodiesterase activity after
ethanol feeding contributed to the inhibition of TNF-
production despite desensitization of cAMP synthesis, measurement of TNF-
production in the presence of Ro 20-1724 should then reflect the decreased cAMP production in Kupffer cells from the ethanol-fed rats.
TNF-
production was inhibited when cells from both pair-fed and
ethanol-fed rats were preincubated with 0.1-10 µM Ro 20-1724 for
1 h before LPS stimulation (Fig. 5).
Treatment with 0.1-10 µM Ro 20-1724 in the absence of LPS had no
effect on TNF-
release (data not shown). TNF-
production did not
differ between ethanol- and pair-fed rats, even in the presence of the
phosphodiesterase inhibitor.
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Activity and quantity of PKA. The
inhibitory effect of cAMP on TNF- production is associated with a
decrease in mRNA for TNF-
in macrophages and Kupffer cells (14, 45).
cAMP-dependent regulation of transcription requires the activation of
PKA and translocation of the catalytic subunit to the nucleus (16). We
therefore compared the activity and quantity of PKA in Kupffer cells
from ethanol and pair-fed rats. Basal and total activity of PKA,
measured in cell homogenates in the absence or presence of 40 µM
dibutyryl-cAMP, did not differ between groups, as measured by the
kemptide assay (Table 4). Similarly, there
was no difference in the quantity of immunoreactive RI, RII, or C
subunits of PKA in homogenates of cultured Kupffer cells from pair-fed
and ethanol-fed rats (Table 4). However, activation of PKA in response
to hormonal stimulation (activity ratio) in intact Kupffer cells was
decreased after ethanol feeding. Treatment of Kupffer cells from
pair-fed rats with 10
6 M
NECA for 15 min resulted in the activation of 42% of total PKA (Table
4). In cells from ethanol-fed rats, the extent of activation was
reduced by 50% compared with the pair-fed group (Table 4), reflecting
the decreased ability of NECA to activate cAMP production after ethanol
feeding.
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As additional measures of hormonal stimulation of PKA activity in
Kupffer cells, the translocation of PKA-C to the nucleus and
phosphorylation of CREB were assessed by immunohistochemistry. In
nonstimulated cells, PKA-C was predominantly localized to extranuclear regions of Kupffer cells isolated from both ethanol- and pair-fed rats
(Fig.
6A).
Translocation of the catalytic subunit was observed after 30 min of
stimulation with 106 M NECA
in both groups (Fig. 6A).
Similarly, the appearance of phospho-CREB in the nucleus was observed
after 30 min of treatment with 10 nM forskolin in cells from
both ethanol- and pair-fed animals (Fig.
6B). Although immunohistochemistry
does not allow for a quantitative comparison between ethanol- and
pair-fed animals, Kupffer cells from both groups responded to hormonal
activation, despite the reduced production of cAMP and percent
activation of PKA after ethanol feeding.
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DISCUSSION |
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Chronic ethanol feeding to rats caused a desensitization of
hormone-stimulated cAMP production in isolated and cultured Kupffer cells. This ethanol-induced desensitization was characterized by a
decrease in the quantity of Gs
protein and decreased receptor-stimulated cAMP production.
Surprisingly, this desensitization of the cAMP signaling cascade did
not interfere with the ability of hormones coupled to
Gs
to inhibit TNF-
production after treatment with LPS or latex beads. Although total PKA
activity was not affected by ethanol feeding, activation of PKA in
response to hormonal stimulation was reduced in Kupffer cells from
ethanol-fed rats compared with controls. However, translocation of
PKA-C to the nucleus and phosphorylation of CREB in response to
hormonal stimulation were observed after ethanol feeding, despite the
profound reduction in cAMP production in response to hormones acting
via Gs
. These data suggest that
ethanol exposure resulted in a dissociation between cAMP production and
the downstream inhibition of TNF-
production mediated by PKA.
The cAMP signal transduction cascade is under complex regulatory control; mechanisms for enhancing or dampening responses to hormonal activation can occur via regulation of cAMP concentration per se or the activity of PKA. Concentration of cAMP in response to activation can be controlled by receptor function and quantity, G protein function and quantity, and activity of adenylyl cyclase and phosphodiesterases. Regulation of activity of PKA can be mediated by changes in subunit expression, activity, and localization (19). Activity of PKA can also be regulated depending on the quantity and localization of the heat-stable inhibitor of PKA (PKI). PKI associates with PKA-C in the nucleus, inhibiting its activity and mediating its export from the nucleus (39, 40). Finally, there is also regulation of the quantity of cAMP-responsive transcription factors, CREB and cAMP response element modulator (CREM) (15).
Chronic ethanol exposure desensitizes G protein-stimulated cAMP
production in a number of cell types (7), similar to the decrease in
hormone-dependent cAMP production found in freshly isolated and
cultured Kupffer cells after chronic ethanol feeding (Figs. 1-2).
Degradation of cAMP by phosphodiesterases does not appear to be a
target for ethanol action; we found no effect of ethanol on activity of
phosphodiesterase in hepatocytes (30), and inhibition of type IV
phosphodiesterase decreased TNF- production to an equivalent extent
in Kupffer cells from ethanol- and pair-fed rats (Fig. 5). Ethanol
feeding had no effect on the total in vitro activity of PKA measured in
Kupffer cell extracts (Table 4). However, activation of PKA in the
intact cell by the adenosine receptor agonist NECA was 50% lower in
Kupffer cells after ethanol feeding compared with controls (Table 4).
cAMP production in response to
10
6 M NECA was only 27% of
control values in Kupffer cells after ethanol feeding (Fig. 2),
indicating that there was amplification of the hormone-dependent signal
in ethanol-fed rats at this point in the cAMP signalling cascade.
Moreover, despite the lower degree of activation of PKA in response to
10
6 M NECA, activation was
sufficient in the Kupffer cells from ethanol-fed rats to detect
translocation of PKA-C to the nucleus within 30 min after stimulation.
In some cell types, ethanol may also regulate the
localization/translocation of PKA-C. In cultured neuroblastoma X glioma hybrid cells (NG108-15), chronic exposure to ethanol during
culture results in the movement of PKA-C to the nucleus independent of the application of exogenous agonist (8). Because PKA-C was not
sequestered to the nucleus in nonstimulated Kupffer cells from
ethanol-fed animals, agonist-independent movement of PKA-C to the
nucleus is not likely to account for the dissociation of cAMP
production from inhibition of TNF- by hormones in our system. Moreover, if PKA-C was localized to the nucleus in the absence of
agonist, we would expect lower TNF-
release in ethanol-fed rats,
even in the absence of agonist. In contrast to this prediction, TNF-
release is higher in ethanol-fed rats in the absence of agonist.
Because the mislocalization of the catalytic subunit is restored in
NG108-15 cells within 9 h of ethanol removal (8), it is possible
that any ethanol-induced changes in localization of PKA-C have
recovered in Kupffer cells from ethanol-fed rats after 24-h culture in
the absence of ethanol.
Another step in the cAMP signal transduction cascade that may be targeted by long-term ethanol feeding is the regulation of transcriptional control by the nuclear transcription factor CREB. However, very little information is available on the potential effects of ethanol on CREB phosphorylation/dephosphorylation, interaction with other nuclear transcription factors, or cAMP response elements. In rat cerebellum, short-term ethanol exposure increases the phosphorylation of CREB, whereas chronic ethanol has no effect (43). In Kupffer cells, ethanol feeding did not impact on the ability of forskolin to increase the level of immunoreactive phospho-CREB in Kupffer cells.
Taken together, these data suggest that ethanol feeding potentiates
responses to cAMP. Despite very low rates of cAMP accumulation after
ethanol feeding, localization of the catalytic subunit to the nucleus
and its ability to phosphorylate CREB in response to stimulation of
cAMP production were maintained. The mechanism for this potentiation is
not known. It may be that chronic ethanol exposure specifically
enhances the amplification of the cAMP signal at one or more points in
the signalling cascade. Activation of PKA by cAMP may be one site of
amplification, since endogenous activation of PKA by
106 M NECA was reduced by
only 50%, whereas cAMP production at this concentration was reduced by
75%. Alternatively, it is possible that no unique amplification
mechanism is required after ethanol feeding, i.e., the normal
mechanisms of signal amplification from cAMP to the downstream response
may be sufficient to amplify the signal from the small amount of second
messenger generated in response to hormonal activation after ethanol feeding.
Kupffer cells, the resident macrophage in the liver, have been
implicated in the development of alcoholic liver injury (1, 26).
Long-term alcohol consumption is associated with an increase in
circulating TNF- (21, 25, 44) and an increased response to endotoxin
treatment (18). Increased TNF-
observed after alcohol consumption
(21, 25, 44) is likely due both to increased exposure to circulating
endotoxins (21, 26) and enhanced Kupffer cell responses to this
activation. When rats fed ethanol for 6 wk are injected with LPS, the
concentration of circulating TNF-
was fivefold greater than in
LPS-treated controls (17). We have found that basal release of TNF-
and the response to low concentrations of LPS were higher in Kupffer
cells isolated from ethanol-fed rats compared with pair-fed controls
(Fig. 3). The mechanism for this enhanced production of TNF-
is not
known. However, our data indicate that increased TNF-
after ethanol
feeding is not due to a loss in the activity of the feedback inhibitory
pathway mediated by cAMP. We have been able to isolate the effects of
ethanol on cAMP-mediated inhibition of TNF-
release and have found
that Kupffer cells from ethanol-fed rats retain the ability to inhibit TNF-
production via cAMP-dependent hormonal activation. Thus the
increased susceptibility to endotoxin-induced liver injury observed
after ethanol feeding (18) is likely the result of an enhanced
stimulatory response of Kupffer cells to activation by endotoxin rather
than an impairment in the inhibitory feedback loop mediated by cAMP.
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ACKNOWLEDGEMENTS |
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This work was supported in part by grants from the Alcoholic Beverage Medical Research Foundation and National Sciences and Engineering Research Council, Canada.
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FOOTNOTES |
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Present address of A. Aldred: Dept. of Health Studies and Gerontology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Address for reprint requests: L. E. Nagy, Dept. of Nutrition, Case Western Reserve Univ., Cleveland, OH 44106-4906.
Received 1 October 1997; accepted in final form 10 September 1998.
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