Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365
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ABSTRACT |
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Hepatic macrophages are
sensitized to alcohol in 24 h due to increases in the endotoxin
receptor, CD14; however, desensitization to lipopolysaccharide (LPS),
which occurred earlier, could not be explained by changes in CD14.
Therefore, the purpose of this work was to attempt to understand
factors responsible for ethanol-induced desensitization to LPS in
hepatic macrophages. Rats were given ethanol (5 g/kg body wt)
intragastrically, and hepatic macrophages were isolated 2 h later.
After addition of endotoxin, intracellular Ca2+
concentration ([Ca2+]i) was
measured using fura 2 and tumor necrosis factor (TNF)- was measured
by ELISA. Ethanol given 2 h before injection of LPS totally prevented
liver injury and blunted LPS-induced increases in
[Ca2+]i and TNF-
in hepatic
macrophages. Furthermore, the protein kinase C (PKC) agonist phorbol
12-myristate 13-acetate and acute ethanol treatment both activated PKC
and largely prevented the influx of
[Ca2+]i caused by LPS.
Sterilization of the gut with antibiotics completely blocked all
effects of ethanol on [Ca2+]i and
TNF-
release. Thus ethanol-induced desensitization of hepatic
macrophages correlates with gut-derived endotoxin after ethanol and
involves the effect of PKC on voltage-dependent Ca2+ channels.
intracellular calcium; tumor necrosis factor-; protein kinase C; lipopolysaccharide
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INTRODUCTION |
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RECENTLY, IT WAS SHOWN that ethanol causes sensitization of rat hepatic macrophages via mechanisms dependent on endotoxin and increases in CD14 (11); however, desensitization after alcohol could not be explained by decreases in CD14. Ethanol attenuates the lipopolysaccharide (LPS)-induced production of various biologically active substances such as proteases, toxic radicals, eicosanoids, and cytokines from hepatic macrophages (4, 9, 19, 21, 29). Moreover, it was previously demonstrated that desensitization of hepatic macrophages was caused by gut-derived endotoxin (11). Reasons for this desensitization to ethanol are not understood.
Membrane depolarization opens voltage-dependent Ca2+
channels, promoting Ca2+ entry and leading to subsequent
Ca2+-dependent signaling events in various cell types (5).
Intracellular Ca2+ concentration
([Ca2+]i) most likely plays an
important role in activation of hepatic macrophages (7, 8). For
example, a transient increase of [Ca2+]i is required for LPS-induced
expression of tumor necrosis factor (TNF)- by a macrophage cell line
(34). Furthermore, hepatic macrophages contain voltage-dependent
Ca2+ channels(15), which most likely play an important role
in modulating hepatic macrophage function by regulating
[Ca2+]i. Paradoxically,
voltage-dependent Ca2+ channels were inactivated by acute
exposure to ethanol (i.e., desensitization) (14) but were easier to
open after chronic alcohol treatment (i.e., sensitization) (12).
However, the mechanisms by which voltage-dependent Ca2+
channels are regulated in hepatic macrophages remain unknown and
whether or not they participate in desensitization to ethanol in vivo
and in vitro is not clear.
Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) are
exogenous compounds that activate protein kinase C (PKC) in a manner
analogous to diacylglycerol, an endogenous product of
phosphatidylinositol breakdown (22). PKC has many regulatory roles,
including cell proliferation and differentiation (17), and has been
shown to modulate ion channels, including Ca2+ channels
(27), by phosphorylating the -subunit of the channel. Although
hepatic macrophages are known to have PKC activity (10), whether PKC
participates in the mechanism of the regulation of voltage-dependent
Ca2+ channels is not clear. Therefore, the present study
was designed to determine if cellular desensitization in hepatic
macrophages due to acute ethanol treatment involves voltage-dependent
Ca2+ channels and PKC.
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MATERIALS AND METHODS |
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Animals and treatments.
Female Sprague-Dawley rats weighing between 200 and 250 g were used in
this study. All animals were given humane care in compliance with
institutional guidelines. Rats were given ethanol (5 g/kg body wt po)
before experiments (31, 36). Some ethanol-treated rats were also
treated for 4 days with polymyxin B and neomycin (26) to prevent growth
of intestinal bacteria, the primary source of endotoxin in the
gastrointestinal tract. On the basis of the results of preliminary
experiments, 150 mg · kg1 · day
1
of polymyxin B and 450 mg · kg
1 · day
1
of neomycin were given orally. Under these conditions, gut
sterilization was achieved (1). Some rats were treated with microcystin
(50 µg/kg ip) 1 h before intragastric injection of ethanol to inhibit dephosphorylation.
Analytical methods.
Rats were forced to breathe into a closed, heated chamber (37°C)
for 20 s, and 1 ml of breath was collected using a gas-tight syringe to
measure ethanol by gas chromatography (31, 36). Blood was collected
from the portal vein in pyrogen-free heparinized syringes and
centrifuged, and plasma was stored at 20°C in pyrogen-free glass test tubes until endotoxin was measured using the Limulus amebocyte lysate assay (Whitaker Bioproducts, Walkerville, MD). Levels
of endotoxin in plasma from normal rats were below the limits of
detection. Serum was stored at
20°C in microtubes, and
aspartate transaminase and alanine aminotransferase were measured by
standard enzymatic procedures (6). Livers were formalin fixed, embedded
in paraffin, and stained with hematoxylin and eosin to assess
inflammation and necrosis.
Hepatic macrophage preparation and culture.
Hepatic macrophages were isolated by collagenase digestion and
differential centrifugation using Percoll (Pharmacia, Uppsala, Sweden)
as described elsewhere, with slight modifications (24). Briefly, the
liver was perfused through the portal vein with Ca2+- and
Mg2+-free Hanks' balanced salt solution (HBSS) at 37°C
for 5 min at a flow rate of 26 ml/min. Subsequently, perfusion was
performed with HBSS containing 0.025% collagenase IV (Sigma Chemical,
St. Louis, MO) at 37°C for 5 min. After the liver was digested, it was excised and cut into small pieces in collagenase buffer. The suspension was filtered through nylon gauze mesh, and the filtrate was
centrifuged at 450 g for 10 min at 4°C. Cell pellets were resuspended in buffer, parenchymal cells were removed by centrifugation at 50 g for 3 min, and the nonparenchymal cell fraction was
washed twice with buffer. Cells were centrifuged on a density cushion of Percoll at 1,000 g for 15 min, and the hepatic macrophage
fraction was collected and washed again with buffer. Viability of
cells, determined by trypan blue exclusion, was >90%, and purity,
which was assessed histologically, was also >90%. Neutrophils and
lymphocytes could be easily differentiated from hepatic macrophages
based on incorporation of latex beads and nuclear shape. These
preparations were virtually free of neutrophils and lymphocytes.
Moreover, there were no differences between control, ethanol, and
antibiotics plus ethanol groups with respect to viability and purity
(Table 1). Cells were
seeded onto 25-mm glass coverslips and cultured in DMEM (GIBCO Life
Technologies, Grand Island, NY) supplemented with 10% fetal bovine
serum (FBS) and antibiotics (100 U/ml of penicillin G and 100 µg/ml
of streptomycin sulfate) at 37°C with 5% CO2.
Nonadherent cells were removed after 1 h by replacing buffer, and cells
were cultured for 24 h before experiments.
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Measurement of
[Ca2+]i.
[Ca2+]i was measured
fluorometrically using the Ca2+ indicator dye fura 2 and a
microspectrofluorometer (PTI, South Brunswick, NJ) interfaced with an
inverted microscope (Diaphot, Nikon). Hepatic macrophages were
incubated in modified Hanks' buffer (in mmol/l: 115 NaCl, 5 KCl, 0.3 Na2HPO4, 0.4 KH2PO4,
5.6 glucose, 0.8 MgSO4, 1.26 CaCl2, and 15 HEPES, pH 7.4) containing 5 µmol/l fura 2-AM (Molecular Probes,
Eugene, OR) and 0.03% Pluronic F-127 (BASF Wyandotte, Wyandotte, MI)
at room temperature for 60 min. Coverslips plated with hepatic
macrophages were rinsed and placed in chambers with buffer at room
temperature. Changes in fluorescence intensity of fura 2 at excitation
wavelengths of 340 and 380 nm and emission at 510 nm were monitored in
individual hepatic macrophages. Each value was corrected by subtracting
the system dark noise and autofluorescence, assessed by quenching fura
2 fluorescence with Mn2+, as described previously (15).
[Ca2+]i was determined from the
following equation: [Ca2+]i = Kd[(R Rmin)/(Rmax
R)]/(Fo/Fs), where
Fo/Fs is the ratio of fluorescent intensities
evoked by 380-nm light from fura 2 pentapotassium salt loaded in cells
using a buffer containing 3 mmol/l EGTA and 1 µmol/l ionomycin
([Ca2+]min) or 10 mmol/l
Ca2+ and 1 µmol/l ionomycin
([Ca2+]max). R is the ratio of
fluorescent intensities at excitation wavelengths of 340 nm and 380 nm,
and Rmax and Rmin are values of R at
[Ca2+]max and
[Ca2+]min, respectively. The values
of these constants were determined at the end of each experiment, and a
dissociation constant (Kd) of 135 nmol/l was used
(13).
TNF- detection.
Hepatic macrophages were seeded onto 24-well plates and cultured in
DMEM supplemented with 10% FBS and antibiotics at 37°C in the
presence of 5% CO2. Cells were incubated with fresh medium containing LPS (0 and 1 µg/ml supplemented with 5% rat serum) for an
additional 4 h. Samples of medium were collected and kept at
80°C until assay. TNF-
in the culture medium was measured using an ELISA kit (Genzyme, Cambridge, MA), and data were corrected for dilution.
[3H]phorbol 12,13-dibutyrate binding to subcellular fractions from hepatic macrophages. Hepatic macrophages from rats treated acutely with ethanol for 2 h were collected and resuspended in homogenizing buffer at a density of ~30 × 106 cells/ml. Cells were then incubated in the dark with either PMA (100 nmol/l, Sigma Chemical) or DMSO vehicle. After 15 min, the reaction was stopped by rinsing cells three times with ice-cold PBS. Cells were then collected using a rubber policeman, homogenized (30-40 times) on ice in a 2-ml homogenizing tube, and centrifuged in an airfuge (50,000 rpm, 1 h, 4°C). The pellet was resuspended in 250 µl of buffer, and the supernatant was concentrated to a final volume of 250 µl (33).
PKC activity for both particulate and soluble fractions was estimated by measuring the binding of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) to the active form of the PKC molecule (37). The reaction mixture contained 46 mmol/l Tris · HCl (pH 7.4), 1 mmol/l CaCl2, 0.1 mg/ml BSA, 0.02 mol/l MgCl2, 2 mmol/l dithiothreitol, 0.04 mg/ml phosphatidylserine, and [3H]PDBu (15.6 mCi · nmolStatistical analysis. All results are expressed as means ± SE. Statistical differences between means were determined using ANOVA or ANOVA on ranks as appropriate. P < 0.05 was selected before the study to reflect significance.
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RESULTS |
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Blood ethanol and endotoxin levels. After oral administration of ethanol (5 g/kg) to untreated, normal rats, blood ethanol levels increased gradually and reached a value of 268 ± 38 mg/dl after 90 min. Similar results were obtained in rats treated with antibiotics (245 ± 34 mg/dl). Subsequently, levels declined toward basal values over the next 6 h. Endotoxin was not detectable in peripheral blood; however, levels were increased in portal blood by ethanol treatment to values of 77 ± 6 pg/ml at 90 min and declined subsequently to baseline. Levels were three- to fourfold significantly higher than values from rats treated with antibiotics (23 ± 1 pg/ml, P < 0.05).
Effect of ethanol on survival after LPS injection.
LPS (10 mg/kg) was injected via the tail vein, and 24-h survival rates
were assessed (Fig. 1). Mortality rates
were ~50% with 10 mg/kg of LPS in control rats, with most rats dying
between 8 and 24 h. However, mortality was prevented totally by ethanol administration (5 g/kg) 2 h before LPS. This phenomenon was abolished by treatment with antibiotics, confirming that endotoxin is involved in
mechanisms of desensitization to endotoxin due to ethanol in the animal
(11).
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Effect of ethanol and LPS on liver histology and serum
transaminases.
Liver specimens were collected for histology 24 h after administration
of LPS (10 mg/kg). Histology was normal in control rats (Fig.
2A), whereas 24 h after LPS
administration focal necrosis and neutrophil infiltration were observed
in the liver as expected (Fig. 2B). Two hours after ethanol
treatment, however, necrosis and infiltration caused by LPS were
decreased (Fig. 2C). Moreover, antibiotics prevented the
improvement in histology observed with ethanol (Fig. 2D).
Similar results were obtained with serum transaminases (Fig.
3). Mean values for aspartate transaminase
and alanine aminotransferase in the control group were 26 ± 3 and 29 ± 4 IU/l, respectively. LPS increased transaminases
dramatically to 823 ± 84 and 730 ± 128 IU/l, respectively. However,
in ethanol-treated rats, this effect was blunted, and protection was
prevented with antibiotics (Fig. 3).
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Effect of ethanol treatment on LPS-induced increases in
[Ca2+]i
in hepatic macrophages.
[Ca2+]i increases both from
Ca2+ release from the endoplasmic reticulum as well as from
influx from the extracellular space via voltage-dependent
Ca2+ channels. Addition of LPS (10 µg/ml) to
hepatic macrophages from untreated control rats caused a transient
elevation in [Ca2+]i, which
consisted of an initial sharp increase followed later by a large
increase and a gradual return to the baseline over 3 min. The maximal
[Ca2+]i level achieved, including
both components, was 304 ± 14 nmol/l (Fig.
4A, Table
2); however, values only reached 116 ± 9 nmol/l and only exhibited the sharp component when extracellular
Ca2+ was removed (Fig. 4B). The area under the
curve of the [Ca2+]i trace was 315 ± 23 s · nmol1 · l
1
for cells from control rats. However, area was reduced ~10-fold in
cells from rats treated with ethanol. This large component of the
[Ca2+]i signal was blocked by a
dihydropyridine-type Ca2+ channel blocker (16). Thus the
large, slower component was due to Ca2+ entry from the
extracellular space via voltage-dependent Ca2+ channels. In
cells isolated from rats treated with ethanol 2 h earlier, the
extracellular component was also absent (Fig. 4C). Thus these
data are consistent with the hypothesis that desensitization due to
alcohol involves downregulation or inactivation of voltage-dependent Ca2+ channels in hepatic macrophages. Treatment of rats
with antibiotics prevented this desensitization, as the large
two-component increase in
[Ca2+]i was restored (Fig.
4E). Antibiotics did not affect the small release of
Ca2+ from intracellular stores (Fig. 4F). Moreover,
the dihydropyridine-type Ca2+ channel blocker,
nimodipine, and the phospholipase C inhibitor, 2-nitro-4-carboxyphenyl
N,N-diphenylcarbamate, largely prevented the increase
in [Ca2+]i in hepatic macrophages
after LPS stimulation as expected (Table 2), indicating that the
LPS-induced [Ca2+]i response is
dependent on voltage-dependent Ca2+ channels via mechanisms
involving phospholipase C. However, the response of hepatic macrophages
isolated from control rats to LPS was not affected by treatment with
antibiotics (data not shown).
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Acute exposure to ethanol increases PKC activity in hepatic
macrophages.
The effect of PMA, the classical PKC agonist, on membrane-bound PKC
levels in cultured hepatic macrophages is depicted in Fig.
5A. Treatment of hepatic
macrophages with this ligand for 15 min significantly increased PKC
activity over control levels around 13-fold. In untreated cells, 62%
of the activity was particulate, whereas 38% was in the soluble
fraction. After exposure to PMA, however, 96% was particulate and 4%
was soluble. Similarly, acute ethanol treatment increased values in the
particulate fraction to 100%. The effect of acute treatment with
ethanol on membrane-bound PKC activity is depicted in Fig. 5A.
PKC activity in hepatic macrophages isolated from rats exposed acutely
to ethanol was increased significantly, around 12-fold compared with
controls (Fig. 5A).
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Role of PKC on LPS-induced increases of
[Ca2+]i
in isolated hepatic macrophages.
Representative changes in [Ca2+]i
in individual cultured hepatic macrophages caused by LPS are depicted
in Fig. 5B. After addition of LPS (10 µg/ml), the maximal
[Ca2+]i level in normal hepatic
macrophages was 304 ± 14 nmol/l (n > 20). In contrast, when
hepatic macrophages were pretreated with PMA (1 µmol/l) 15 min before
the addition of LPS, the increase in
[Ca2+]i only reached a value of 106 nmol/l in this example (mean = 163 ± 31 nmol/l, P < 0.05).
The area under curve of the [Ca2+]i
trace was 315 ± 23 s · nmol1 · l
1
in cells from control rats; however, values only reached 43 ± 9 s · nmol
1 · l
1
in hepatic macrophages pretreated with PMA. PMA alone had no effect on
the basal level of [Ca2+]i.
Importantly, only the sharp transient peak, similar to what was seen
with ethanol (Fig. 4), was observed.
Phosphatase inhibitor microcystin blocks desensitization to ethanol
in isolated hepatic macrophages.
After addition of LPS (10 µg/ml), the maximal
[Ca2+]i level in hepatic
macrophages treated with ethanol achieved was 116 ± 9 nmol/l (Fig.
6A). In contrast, when rats were
pretreated with 50 µg/kg ip of the phosphatase inhibitor microcystin
1 h before intragastric injection of ethanol, the increase in
[Ca2+]i was restored to 300 ± 10 nmol/l. Microcystin alone had no effect on the LPS-induced
[Ca2+]i response.
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Effect of ethanol treatment in vivo on LPS-induced production of
TNF- by isolated hepatic macrophages.
To evaluate the effect of ethanol on cytokine production by hepatic
macrophages, LPS-induced TNF-
production was measured after ethanol
treatment (Fig. 6B). As expected, isolated hepatic macrophages
produced large amounts of TNF-
in the presence of LPS (300% of
basal values). However, ethanol completely prevented this effect.
Moreover, treatment of rats with antibiotics or microcystin prevented
the decrease in TNF-
production caused by exposure to ethanol
for 2 h.
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DISCUSSION |
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Mechanism of sensitization to endotoxin by ethanol.
TNF- levels increase in alcoholics, consistent with the hypothesis
that hepatic macrophages of patients with alcoholic liver disease are
activated. Shibayama et al. (28) demonstrated that acute administration
of ethanol enhances endotoxin hepatotoxicity. Moreover, it was shown
recently that hepatic macrophages became sensitized to LPS in cells
isolated 24 h after ethanol, reflected by increased
[Ca2+]i, TNF-
production, and
large increases in the endotoxin receptor, CD14 (11). These effects
were all blocked by antibiotics, indicating that sensitization of
hepatic macrophages by ethanol is also mediated by gut-derived
endotoxin. Moreover, hepatic macrophages from rats treated with ethanol
24 h earlier expressed much higher levels of CD14 than rats given
antibiotics. This most likely explains why hepatic macrophages ex vivo
"remember" that they were treated with ethanol in vivo. Because
gut sterilization removes portal endotoxin, hepatic macrophages are not
sensitized and liver damage after LPS injection is blunted in
vivo (Figs. 2 and 3). Recently, Su et al. (30) showed that CD14 was
upregulated in hepatic macrophages from rats exposed to chronic alcohol
using the Tsukamoto-French enteral ethanol delivery model.
Endotoxin is involved in desensitization to LPS caused by ethanol.
Endotoxin shock causes cardiovascular collapse, lung dysfunction, and
altered blood coagulation, and hepatic macrophage-derived cytokines
play an important role in this phenomenon (32). Accordingly, the
LPS-induced increase of [Ca2+]i and
TNF- production was measured in hepatic macrophages. Previous studies have shown that acute alcohol decreases endotoxin-induced increases in cytotoxic mediators such as TNF-
(9, 21), superoxide (4), and nitric oxide (29). Furthermore, it was demonstrated that acute
ethanol protects against otherwise lethal doses of LPS. Endotoxin is
involved in this phenomenon,since sterilization of the gut with
antibiotics blocked this effect (Figs. 1-3).
Hepatic macrophages are involved in ethanol-induced desensitization
in vivo.
Hepatic macrophages represent more than 80% of fixed tissue
macrophages (19) and play an important role in removal of gut-derived endotoxin (LPS) (23). Moreover, hepatic macrophages are a rich source
of cytokines after LPS treatment (3). LPS elevates
[Ca2+]i transiently in isolated
hepatic macrophages (Fig. 4). Most likely, Ca2+ enters from
the extracellular space via voltage-dependent Ca2+ channels
because the LPS-induced increase in
[Ca2+]i was largely blocked by the
Ca2+ channel blocker, nimodipine (16) (Table 2).
Nisoldipine, a related Ca2+ channel blocker, also inhibited
the production of TNF- due to LPS in cultured hepatic macrophages
(25). Therefore, it is concluded that the transient increase in
[Ca2+]i due to LPS is important in
triggering the production of TNF-
by hepatic macrophages. As
expected, ethanol prevented the increase in
[Ca2+]i due to LPS (Fig. 4). In
this model, increases in TNF-
production from hepatic macrophages
were also markedly blunted by ethanol (Fig. 6B). Importantly,
sterilization of the gut with antibiotics blocked all effects of
ethanol on [Ca2+]i and TNF-
production completely (Figs. 4 and 6), supporting the hypothesis that
desensitization in the hepatic macrophages due to alcohol involves
gut-derived endotoxin (4).
Voltage-dependent Ca2+
channels in hepatic macrophages are involved in desensitization to LPS
caused by ethanol.
Hepatic macrophages isolated from rats exposed to ethanol for 2 h were
much less sensitive to LPS, as reflected by blunted [Ca2+]i and TNF- production
(Figs. 4 and 6). However, the LPS receptor CD14 was not decreased 2 h
after ethanol treatment when the response of the hepatic macrophages to
endotoxin was clearly blunted (11). However, Hijioka et al. (14) showed
that acute ethanol treatment in vivo reduced the probability that
L-type voltage-dependent Ca2+ channels in hepatic
macrophages would be open. Increases in intracellular Ca2+
after addition of LPS depend on both Ca2+ release from the
endoplasmic reticulum and influx from the extracellular space via
voltage-dependent Ca2+ channels. When LPS was added, a
sharp increase, followed later by a large increase and a gradual return
to the baseline, was observed; however, only the sharp component
occurred when extracellular Ca2+ was removed (Fig.
4B). Thus it is concluded that the large, slower component
represents Ca2+ entry from the extracellular space via
voltage-dependent Ca2+ channels. In cells isolated from
rats treated for 2 h with ethanol, the extracellular component was
absent (Fig. 4C). Thus it is concluded that desensitization due
to alcohol involves inactivation of voltage-dependent Ca2+
channels in hepatic macrophages. Furthermore, it is concluded that
endotoxin is involved in this phenomenon, since treatment of rats with
antibiotics prevented this desensitization (Figs. 4E and
6B).
Role of PKC.
After LPS challenge of murine peritoneal macrophages, production of
TNF- was inhibited by the PKC inhibitor H-7 in a dose-dependent manner (18). Furthermore, PKC was upregulated in mouse hepatic macrophages treated with PMA, and more TNF-
was produced after LPS
stimulation in these cells than in untreated cells (2). However,
superoxide production by LPS-treated hepatic macrophages was suppressed
by PMA, and PGE2 release by LPS/IFN-
-treated hepatic macrophages was increased by treatment with staurosporine (35). Thus
the role of PKC in hepatic macrophage activation remains unclear. In
this study, PKC activity in hepatic macrophages was increased not only
by PMA treatment but by acute ethanol treatment. In both cases,
voltage-dependent Ca2+ channels were inactivated (Fig.
5A). The LPS-induced
[Ca2+]i response was decreased by
treatment with PMA and acute ethanol treatment. The shape of the
[Ca2+]i response by treatment with
PMA was very similar to acute ethanol treatment. Together, these data
strongly support the hypothesis that ethanol-induced increases in
endotoxin activate PKC, which inhibits voltage-dependent
Ca2+ channels in hepatic macrophages leading to cellular
desensitization. Moreover, it is likely that PKC phosphorylates the
-subunit of voltage-dependent Ca2+ channels. This is
based on the finding that inhibition of dephosphorylation by the
phosphatase inhibitor, microcystin, reversed the effect of ethanol on
[Ca2+]i and TNF-
production.
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ACKNOWLEDGEMENTS |
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This work was supported, in part, by grants from National Institute on Alcohol Abuse and Alcoholism.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. G. Thurman, Laboratory of Hepatobiology and Toxicology, Dept. of Pharmacology, CB#7365, Mary Ellen Jones Building, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365.
Received 17 July 1998; accepted in final form 31 August 1999.
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