Kupffer cell-derived prostaglandin E2 is involved
in alcohol-induced fat accumulation in rat liver
Nobuyuki
Enomoto1,
Kenichi
Ikejima1,2,
Shunhei
Yamashina1,
Ayako
Enomoto2,
Teruhiro
Nishiura2,
Tetsuro
Nishimura2,
David A.
Brenner2,
Peter
Schemmer1,
Blair U.
Bradford1,
Chantal A.
Rivera1,
Zhi
Zhong1, and
Ronald G.
Thurman1
1 Laboratory of Hepatobiology and Toxicology, Department of
Pharmacology, and 2 Division of Digestive Disease and Nutrition,
Department of Medicine, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599-7365
 |
ABSTRACT |
Destruction
of Kupffer cells with gadolinium chloride (GdCl3) and
intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of
ethanol-induced fatty liver remain unclear. Accordingly, the role of
Kupffer cells in ethanol-induced fat accumulation was studied. Rats
were given ethanol (5 g/kg body wt) intragastrically, and tissue
triglycerides were measured enzymatically. Kupffer cells were isolated
0-24 h after ethanol, and PGE2 production was measured
by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by
RT-PCR. As expected, ethanol increased liver triglycerides about
threefold. This increase was blunted by antibiotics, GdCl3,
the dihydropyridine-type Ca2+ channel blocker nimodipine,
and the COX inhibitor indomethacin. Ethanol also increased
PGE2 production by Kupffer cells about threefold. This
increase was also blunted significantly by antibiotics, nimodipine, and
indomethacin. Furthermore, tissue triglycerides were increased about
threefold by PGE2 treatment in vivo as well as by a
PGE2 EP2/EP4 receptor agonist,
whereas an EP1/EP3 agonist had no effect.
Moreover, permeable cAMP analogs also increased triglyceride content in
the liver significantly. We conclude that PGE2 derived from
Kupffer cells, which are activated by ethanol, interacts with
prostanoid receptors on hepatocytes to increase cAMP, which causes
triglyceride accumulation in the liver. This mechanism is one of many
involved in fatty liver caused by ethanol.
fatty liver; triglyceride; adenosine 3',5'-cyclic monophosphate; ethanol
 |
INTRODUCTION |
ALTHOUGH ALCOHOL IS
A well-known hepatotoxin, the mechanisms of pathology still
remain unclear. Specifically, the role of lipid accumulation remains
controversial. A single large dose of ethanol in rats causes a
pronounced increase in liver triglycerides that is maximal in ~24 h
and disappears after 48 h (4, 20). Although fatty liver clearly occurs, whether it is a causal event in
ethanol-induced hepatitis and hepatic fibrosis still remains unclear.
Interestingly, inactivation of Kupffer cells with gadolinium chloride
(GdCl3) or decreasing gut-derived endotoxin by diminishing endotoxin by intestinal sterilization with antibiotics (polymyxin B and
neomycin) decreased ethanol-induced steatosis in the enteral feeding
model of Tsukamoto and French (1, 2). These
treatments also prevented early ethanol-induced liver injury
characterized by necrosis and focal inflammation. Moreover, similar
phenomena were observed in rats treated with glycine, which inhibits
Kupffer cells via activation of a glycine-gated chloride channel, and nimodipine, a dihydropyridine-type Ca2+ channel blocker
(15-18).
Therefore, it was hypothesized that activated Kupffer cells are somehow
involved in mechanisms of ethanol-induced fatty liver. Accordingly, the
aim of this study was to attempt to understand if Kupffer cells are
indeed involved in hepatic fat accumulation, and if so, how.
 |
MATERIALS AND METHODS |
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 (33, 34). Twenty-four
rats were treated for 4 days with polymyxin B and neomycin
(32) to prevent growth of intestinal bacteria, the main
source of endotoxin in the gastrointestinal tract. On the basis of the
results of preliminary experiments (2), polymyxin B (150 mg · kg
1 · day
1) and neomycin
(450 mg · kg
1 · day
1) were
given orally to achieve gut sterilization. Twenty-four rats were
also treated with GdCl3, a selective Kupffer cell toxicant, to inactivate Kupffer cells. In this experiment, a single dose of
GdCl3 (10 mg/kg) dissolved in acidic saline was
administered intravenously to rats 24 h before ethanol treatment.
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. Blood ethanol concentration
was assessed from breath ethanol (33, 34).
Blood was collected from the portal vein in pyrogen-free heparinized syringes and centrifuged, and the plasma was stored at
20°C in pyrogen-free glass test tubes until endotoxin was measured using the
Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). Livers were formalin fixed, embedded in paraffin, and stained with
hematoxylin and eosin to assess steatosis (24). Pathology was assessed in a blinded manner by one of the authors and by an
independent pathologist with expertise in rodent liver.
Assay for hepatic triglycerides.
To assess triglyceride content, liver tissue was homogenized in an
equal volume of normal saline and extracted with a mixture of
chloroform and methanol (2:1) as described previously (4, 6, 13). Zeolite was added to remove
phospholipids. The resulting extract was dried under nitrogen and
dissolved in Plasmanate (1 ml), and triglycerides were measured
enzymatically (4-6).
Kupffer cell preparation and culture.
Kupffer cells were isolated by collagenase digestion and differential
centrifugation using Percoll (Pharmacia, Uppsala, Sweden) as described
previously (27) with slight modifications. 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. Subsequent perfusion was 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 Kupffer cell fraction
was collected and washed with buffer again. Viability of cells
determined by trypan blue exclusion was >90%. Cells were seeded onto
24-well culture plates and cultured in RPMI 1640 (GIBCO Laboratories
Life Technologies, Grand Island, NY) supplemented with 10% fetal
bovine serum and 10 mmol/l HEPES and antibiotics (100 U/ml penicillin G
and 100 µg/ml streptomycin sulfate) at 37°C with 5%
CO2. Nonadherent cells were removed after 15 min by
replacing buffer, and cells were cultured for 4 h before experiments.
Measurement of PGE2 in conditioned media from
cultured Kupffer cells.
Kupffer cells isolated from rats were kept in primary culture for
4 h, and supernatants were analyzed for PGE2 by
competitive RIA using 125I-labeled PGE2 from
Advanced Magnetics (Cambridge, MA). Although this antibody reacts with
PGE1, there is <2% cross-reactivity with other
prostaglandins, arachidonic acid, and thromboxane.
RNA preparation and RT-PCR for inducible cyclooxygenase mRNA.
Total RNA was prepared by guanidium/CsCl centrifugation as described
previously (7, 23, 25,
26). The integrity and concentration of RNA was determined
by measuring absorbance at 260 nm followed by electrophoresis on
agarose gels. First-strand cDNA was transcribed from 1 µg RNA using
Moloney murine leukemia virus RT (Life Technologies, Gaithersburg, MD)
and an oligo(dT)16 primer (Perkin Elmer), and PCR was
performed using a GeneAmp PCR system 9600 (Perkin Elmer, Foster City,
CA). The primer sets used in this study are shown in Table
1 (12). We amplified 1 µl of cDNA in a 50 µl reaction buffer containing 10 pmol of forward and
reverse primers, 2.5 U Taq DNA polymerase, 250 mM
2'-deoxynucleoside 5'-triphosphates (dNTPs), and 1× PCR buffer (Perkin
Elmer). The reaction mixture without enzyme and dNTPs was heated at
100°C for 4 min, then a mixture of Taq polymerase and dNTP
was added at 80°C. Thereafter, 40 cycles of denaturing at 94°C for
60 s, annealing at 50°C for 90 s, and extension at 72°C
for 120 s followed by final extension at 72°C for 10 min were
carried out. The size of the PCR products was verified by
electrophoresis in 2% agarose gels followed by ethidium bromide
staining. Densitometric analysis using NIH image software was performed
for semiquantification of PCR products.
Tissue extraction.
One part of the liver was homogenized with 9 parts 10% TCA using a
Polytron homogenizer. The supernatant was centrifuged with 5 vol of
water-saturated ether. The ether layer then was removed, and extraction
of the aqueous layer was repeated two times. Residual ether was removed
from the aqueous layer.
Measurement of cAMP.
Intracellular cAMP was measured in suspensions of parenchymal cells by
RIA using 125I-labeled-cAMP from Biomedical Technologies
(31). Parenchymal cells were incubated in RPMI 1640 medium
containing various concentrations of PGE2 at 37°C. For
some experiments, 0.5 mM IBMX was preincubated with parenchymal cells
for 2 min before the addition of PGE2. After 5 min, cells
were washed with cold PBS, centrifuged in polypropylene tubes, and
treated with 0.05 M HCl. Tubes were then placed in boiling water for 3 min. Standards and unknowns were combined with tracer solution and
antibody and were incubated 18-20 h at 4°C. Acetate buffer (1 ml) was added, the tubes were centrifuged, and the pellets were
separated from the supernatant. Radioactivity in the precipitate was
counted and compared with known values from a standard curve.
Statistical analysis.
All results were 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.
 |
RESULTS |
Effect of antibiotics.
After oral administration of ethanol (5 g/kg) to untreated normal rats,
blood ethanol levels increased gradually and reached ~250 mg/dl after
90 min (Table 2). Similar results were
obtained in rats treated with antibiotics. Plasma endotoxin levels in
the portal vein were increased about fourfold to ~90 pg/ml 1.5 h
after ethanol (Table 2). This effect was blocked by antibiotics.
Effect of drugs affecting Kupffer cells on hepatic triglyceride
levels and liver histology.
Liver weight was measured before experiments, and no significant
differences between the groups studied were detected (data not shown).
Liver specimens were collected for histology 24 h after
administration of ethanol (5g/kg po). Histology was normal in control
rats (Fig. 1A), whereas
ethanol caused steatosis in the liver as expected (Fig. 1B).
Histological changes were blunted almost completely by intestinal
sterilization with antibiotics, inactivation of Kupffer cells with
GdCl3, inhibition of intracellular Ca2+
concentration influx with the Ca2+ channel blocker
nimodipine, and the cyclooxygenase (COX) inhibitor indomethacin (Fig.
1, C-F).

View larger version (142K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of drugs affecting Kupffer cells on changes in
liver histology due to ethanol. Photomicrographs (hematoxylin and
eosin) are of livers from rats treated as described in MATERIALS
AND METHODS. Nimodipine, a dihydropyridine-type Ca2+
channel blocker, was given to prevent the increase in intracellular
Ca2+ concentration in Kupffer cells after
lipopolysaccharide (LPS) addition (16). A single dose of
nimodipine (1 mg/kg ip) was administered 1 h before ethanol.
Indomethacin, a cyclooxygenase (COX) inhibitor, prevented
PGE2 production in Kupffer cells after LPS stimulation. On
the basis of the results from preliminary experiments, indomethacin (3 mg · kg 1 · day 1) was given
daily 7 days before ethanol treatment. A: no treatment;
B: 24 h after ethanol (5 g/kg po); C:
antibiotics for 4 days and ethanol for 24 h; D:
gadolinium chloride (GdCl3) for 24 h and ethanol for
24 h; E: nimodipine for 1 h and ethanol for
24 h; F: indomethacin (3.6 mg · kg 1 · day 1) for 7 days and ethanol
for 24 h (original magnification, ×400); n = 4 typical photomicrographs.
|
|
Mean liver triglycerides in vehicle-treated controls were 5.5 ± 1.0 mg/g liver (Fig. 2), and values were
increased about threefold by ethanol as expected. This increase was
prevented nearly completely by prior treatment of rats with
antibiotics, GdCl3, nimodipine, or indomethacin.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of drugs affecting Kupffer cells on hepatic
triglyceride levels. Rats were treated with ethanol as described in
MATERIALS AND METHODS. Hepatic triglycerides were extracted
and measured enzymatically. Results are means ± SE;
n = 4. * P < 0.05 vs. control;
# P < 0.05 vs. ethanol by ANOVA and Bonferroni's
post hoc test.
|
|
PGE2 production by conditioned media from Kupffer cells
treated with ethanol.
PGE2 production by Kupffer cells from untreated control
rats was 43 ± 5 pmol · 106
cells
1 · 4 h
1 (Fig.
3). Ethanol treatment increased values
about threefold. This increase was also blunted significantly by
antibiotics, nimodipine, or indomethacin.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of drugs affecting Kupffer cells on
PGE2 production by conditioned media from Kupffer cells.
Rats were treated with ethanol and drugs affecting Kupffer cells as
described in MATERIALS AND METHODS and the Fig. 1 legend.
PGE2 production by conditioned media from Kupffer cells was
measured by ELISA. Results are means ± SE; n = 4. * P < 0.05 vs. control; # P < 0.05 vs. ethanol by ANOVA and Bonferroni's post hoc test.
|
|
Effect of PGE2, ethanol, and cAMP analogs on tissue
triglycerides.
Liver specimens were collected for histology and measurement of tissue
triglycerides 24 h after injection of PGE2 (1 mg/kg iv). Tissue triglycerides were elevated nearly twofold by
PGE2 or ethanol treatment (Fig.
4). Interestingly, ethanol and
PGE2 were additive under these conditions.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of PGE2 on hepatic triglyceride
content. Rats were given prostaglandin E2 (1 mg/kg, iv)
24 h before ethanol. Hepatic triglycerides were extracted and
measured enzymatically as described in MATERIALS AND
METHODS. Results are means ± SE; n = 4. * P < 0.05 vs. control;
# P < 0.05 vs. PGE2 by
ANOVA and Bonferroni's post hoc test.
|
|
Analogs of PGE2, 17-phenyl-omega-trinor-PGE2
(17-PGE2, an EP1/EP3 agonist) or
11-deoxy PGE1 (11-PGE1, an
EP2/EP4 agonist), were injected (1 mg/kg iv)
24 h before tissue triglyceride measurements. Cell permeable cAMP
analogs were also examined for their effect on tissue triglycerides.
Dibutyryl cAMP (DBcAMP; 1 mg/kg) and 8-bromoadenosine cAMP (8-BrcAMP; 1 mg/kg) were injected intravenously 8 h before tissue triglyceride
measurements. The level of cAMP in the liver was increased
significantly by these treatments (Table 3). Moreover, tissue triglycerides were
elevated about threefold by an EP2/EP4 agonist,
but an EP1/EP3 agonist had no effect (Fig. 5). CAMP analogs also increased
triglycerides two- to threefold.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of PGE2 and cAMP analogs on hepatic
triglyceride content. Agonists of PGE2,
17-phenyl-omega-trinor-PGE2 (17-PGE2, an
EP1/EP3 agonist), or 11-deoxy PGE1
(11-PGE1, an EP2/EP4 agonist), were
injected (1 mg/kg iv) 24 h before tissue triglyceride
measurements. Cell permeable cAMP analogs were also examined for their
effect in triglycerides. Dibutyryl-cAMP (DBcAMP; 1 mg/kg) and
8-bromoadenosine cAMP (8-BrcAMP; 1 mg/kg) were injected intravenously
for 8 h before tissue triglyceride measurements. Hepatic
triglycerides were extracted and measured enzymatically as described in
MATERIALS AND METHODS. Results are means ± SE;
n = 4. * P < 0.05 vs. control by
ANOVA and Bonferroni's post hoc test.
|
|
Effect of drugs affecting Kupffer cells on expression of inducible
COX mRNA from rat liver treated with ethanol.
Inducible COX (COX-2) mRNA expression was undetectable in livers from
untreated control rats but was detected within 90 min after
lipopolysaccharide (LPS; 1 mg/kg). Moreover, COX-2 mRNA expression was
increased by treatment with ethanol (2 h) nearly as much as with LPS
(Fig. 6). This increase was blocked
totally by antibiotics, GdCl3, nimodipine, and
indomethacin.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
RT-PCR analysis of inducible COX (COX-2) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. A:
total liver RNA isolated 2 h after ethanol (5 g/kg) administration
was used to detect COX-2 mRNA. GAPDH was also detected as a
housekeeping gene and X174/Hae III was used to
determine the size of PCR products. Lane 1, LPS-treated
positive control; lane 2, untreated control; lane
3, ethanol; lane 4, antibiotics + ethanol;
lane 5, GdCl3 + ethanol; lane 6,
nimodipine + ethanol; lane 7, indomethacin + ethanol; lane 8, no RNA. Gel is representative of 4 individual experiments. MW, molecular weight. B: results are
means ± SE; n = 4. * P < 0.05 vs. control. # P < 0.05 vs. ethanol by ANOVA and
Bonferroni's post hoc test.
|
|
 |
DISCUSSION |
Kupffer cells And endotoxin are involved in mechanisms of fatty
liver.
Many physiological factors participate in ethanol-induced fatty liver
(22). For example, fatty acid synthesis increases, fatty
acid oxidation decreases, release of lipoproteins diminishes, and
systemic adrenergic activity increases cause peripheral lipolysis. With
this study, a role for Kupffer cells can be added to this list. One
possible explanation for the results observed here is as follows. A
single large dose of ethanol increases gut-derived endotoxin in the
circulation (Table 2). Endotoxin is removed from the circulation
primarily by Kupffer cells, which are activated leading to rapid
increases in intracellular Ca2+, which in turn activates
phospholipase A2. This increases PGE2 synthesis
via mechanisms involving COX-2 (21) (Fig. 6).
PGE2 then acts on receptors on hepatocytes to increase
triglycerides in the liver (Fig. 7).
Previous studies (1, 2) showed that inactivation of Kupffer cells with GdCl3 and intestinal
sterilization with antibiotics (polymyxin B and neomycin) prevented
alcohol-induced steatosis in the enteral feeding model of Tsukamoto and
French. However, how the Kupffer cell is involved in mechanisms of
hepatic triglyceride accumulation remains unclear. Here, one single
large dose of ethanol also increased neutral lipid in the liver
(4, 20) (Figs. 1 and 2). Moreover, in this
study, inactivation of Kupffer cells with GdCl3, intestinal
sterilization with antibiotics, prevention of influx of extracellular
Ca2+ with a Ca2+ channel blocker, and
inhibition of COX all reduced hepatic lipid accumulation (Figs. 1 and
2). Accordingly, it is concluded that Kupffer cells and endotoxin are
involved in mechanisms of fatty liver.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 7.
Working hypothesis depicting one mechanism by which
alcohol causes hepatic steatosis via mechanisms dependent on endotoxin
and Kupffer cells. One large dose of ethanol increases gut-derived
endotoxin in the circulation. Endotoxin is removed from the circulation
primarily by Kupffer cells, which are activated leading to rapid
increases in intracellular Ca2+, which in turn activates
phospholipase A2 (PLA2). This increases
PGE2 synthesis via mechanisms involving COX-2.
PGE2 then acts on receptors on hepatocytes to increase
triglyceride accumulation via mechanisms involving cAMP. This presents
only one mechanism involved in fatty liver caused by ethanol. VDCC,
voltage-dependent Ca2+ channel, AA, arachidonic acid, AC,
adenylate cyclase; Gp, G protein, GP, -glycerophosphate, TG,
triglyceride.
|
|
PGE2 is involved in triglyceride accumulation in
hepatocytes.
Eicosanoids are bioactive lipids produced in large quantities by
macrophages from arachidonic acid that is released from membrane lipids
through the action of phospholipase A2 (3,
9). Arachidonic acid produced via the COX pathway leads to
the formation of prostaglandins, key mediators of cell signaling
between Kupffer cells and hepatocytes (28).
PGE2 production from Kupffer cells was enhanced by ethanol, a phenomenon blunted by antibiotics, nimodipine, and indomethacin (Fig.
3). Triglyceride accumulation was also increased by PGE2, and this effect was enhanced by the addition of ethanol (Fig. 4). Many
of the known biological effects of PGE2 are mediated through interaction of PGE2 with specific receptors
(8, 14), and at least four subtypes
(EP1, EP2, EP3, and
EP4) have been characterized pharmacologically and cloned
from at least one species (8, 19). The
specific receptor subtypes are known to be coupled to different signal
transduction pathways. EP1 receptors are coupled to
inositol phospholipid turnover, resulting in an increase of intracellular Ca2+ concentration.
EP2/EP4 receptors act via Gs
proteins and increase cAMP, whereas EP3 receptors are
coupled to Gi and decrease cAMP (28). In this
study, 11-deoxy PGE1, an EP2/EP4
agonist, enhanced triglyceride production, whereas 17-PGE2,
an EP1/EP3 agonist, had no effect on tissue
triglyceride levels (Fig. 5). Moreover, cAMP increased triglyceride
accumulation (Fig. 5). Accordingly, it is concluded that Kupffer
cell-derived PGE2 is involved in triglyceride accumulation
in hepatocytes via mechanisms dependent on hepatocyte
EP2/EP4 receptors and cAMP. Indeed, it has been reported that ethanol increases cAMP, and it is well known that PGE2 increases cAMP (20).
PGE2 is regulated by COX-2.
PGE2 is synthesized in Kupffer cells via the COX pathway
(29, 30), and the COX-2 gene may play an
important role in liver injury. Dinchuk et al. (10) showed
that COX-2 mediates endotoxin-induced liver injury in experiments with
COX-2-deficient mice. On the other hand, indomethacin, a nonspecific
COX inhibitor, prevented histological changes in the liver and
PGE2 production from Kupffer cells caused by gut-derived
endotoxin (Figs. 1-3). Recently, Nanji et al. (25)
showed that upregulation of COX-2 in chronic alcoholic liver injury
increased synthesis of inflammatory and vasoactive eicosanoids.
Furthermore, Nanji et al. (26) showed that dietary saturated fatty acids suppressed COX-2 expression in alcohol-induced liver injury. In this study, COX-2 mRNA expression was increased by
treatment with ethanol in only 2 h (Fig. 6). This expression was
totally blocked by treatment with antibiotics (Fig. 6). Therefore, these data support the hypothesis that endotoxin-induced increases in
COX-2 expression increase PGE2 production.
In summary, one large dose of ethanol is sufficient to increase
gut-derived endotoxin in the circulation. Endotoxin is removed primarily by Kupffer cells that are activated, leading to rapid increases in COX-2 and intracellular Ca2+, the latter of
which in turn activates phospholipase A2. This increases
PGE2, which acts on receptors in hepatocytes to increase accumulation of triglycerides. This pathway is one of many
physiological processes involved in mechanisms of fatty liver caused by
ethanol (Fig. 7).
 |
ACKNOWLEDGEMENTS |
This study was supported in part by grants from the National
Institute on Alcohol Abuse and Alcoholism.
 |
FOOTNOTES |
Portions of this work have been presented previously in abstract form
(see Ref. 11).
Address for reprint requests and other correspondence:
Blair U. Bradford, Laboratory of Hepatobiology and Toxicology,
Dept. of Pharmacology, CB#7365, Mary Ellen Jones Bldg, Univ. of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365 (E-mail: beub{at}med.unc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 26 April 1999; accepted in final form 9 March 2000.
 |
REFERENCES |
1.
Adachi, Y,
Bradford BU,
Gao W,
Bojes HK,
and
Thurman RG.
Inactivation of Kupffer cells prevents early alcohol-induced liver injury.
Hepatology
20:
453-460,
1994[ISI][Medline].
2.
Adachi, Y,
Moore LE,
Bradford BU,
Gao W,
and
Thurman RG.
Antibiotics prevent liver injury in rats following long-term exposure to ethanol.
Gastroenterology
108:
218-224,
1995[ISI][Medline].
3.
Birmelin, M,
and
Decker K.
Ca2+ flux as an initial event in phagocytosis by rat Kupffer cells.
Eur J Biochem
131:
539-543,
1983[Abstract].
4.
Brodie, BB,
Butler WM,
Horning MG,
Maickel RP,
and
Maling HM.
Alcohol-induced triglyceride deposition in liver through derangement of fat transport.
Am J Clin Nutr
9:
432-435,
1961[ISI].
5.
Bucolo, G,
and
David H.
Quantitative determination of serum triglycerides by the use of enzymes.
Clin Chem
19:
476-482,
1973[Abstract/Free Full Text].
6.
Butler, WM,
Maling HM,
Horning MG,
and
Brodie BB.
The direct determination of liver triglycerides.
J Lipid Res
2:
95-96,
1961[Free Full Text].
7.
Chirgwin, JM,
Przybyla AE,
MacDonald RJ,
and
Rutter WJ.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:
5294-5299,
1979[ISI][Medline].
8.
Coleman, RA,
Kennedy I,
Humphrey PPA,
Bunce K,
and
Lumley P.
Prostanoids and their receptors.
In: Comprehensive Medicinal Chemistry, edited by Hansch C,
Sammes PG,
Taylor JB,
and Emmett JC.. Oxford: Pergamon, 1989, p. 643-714.
9.
Decker, K.
Biologically active products of stimulated liver macrophages (Kupffer cells).
Eur J Biochem
192:
245-261,
1990[ISI][Medline].
10.
Dinchuk, JE,
Car BD,
Focht RJ,
Johnston JF,
Jaffee BD,
Covington MB,
Contel NR,
Eng VM,
Collins RJ,
Czerniak PM,
Gorry SA,
and
Trzaskos JM.
Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II.
Nature
378:
406-409,
1995[ISI][Medline].
11.
Enomoto, N,
Ikejima K,
Bradford BU,
Rivera CA,
Arteel GE,
Zhong Z,
and
Thurman RG.
Kupffer cell-derived prostaglandin E2 is involved in alcohol-induced fatty liver (Abstract).
Gastroenterology
114:
1238,
1998.
12.
Feng, L,
Sun W,
Xia Y,
Tang W,
Chanmugan X,
Sayoola E,
Wilson C,
and
Hwang D.
Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression.
Arch Biochem Biophys
307:
361-368,
1993[ISI][Medline].
13.
Folch, J,
Lees M,
and
Stone-Stanley GH.
A simple method for the isolation and purification of total lipid from animal tissue.
J Biol Chem
226:
497-509,
1957[Free Full Text].
14.
Funk, C,
Furci L,
Fitzgerald GA,
Grygorczyk R,
Rochette C,
Bayne MA,
Abramovitz M,
Adam M,
and
Metters MK.
Cloning and expression of a cDNA for human prostaglandin E receptor EP1 subtype.
J Biol Chem
268:
26767-26772,
1993[Abstract/Free Full Text].
15.
Iimuro, Y,
Bradford BU,
Forman DT,
and
Thurman RG.
Glycine prevents alcohol-induced liver injury by decreasing alcohol in the stomach.
Gastroenterology
110:
1536-1542,
1996[ISI][Medline].
16.
Iimuro, Y,
Ikejima K,
Rose ML,
Bradford BU,
and
Thurman RG.
Nimodipine, a dihydropyridine-type calcium channel blocker, prevents alcoholic hepatitis due to chronic intragastric ethanol exposure in the rat.
Hepatology
24:
391-397,
1996[ISI][Medline].
17.
Ikejima, K,
Iimuro Y,
Forman DT,
and
Thurman RG.
A diet containing glycine improves survival in endotoxin shock in the rat.
Am J Physiol Gastrointest Liver Physiol
271:
G97-G103,
1996[Abstract/Free Full Text].
18.
Ikejima, K,
Qu W,
Stachlewitz RF,
and
Thurman RG.
Kupffer cells contain a glycine-gated chloride channel.
Am J Physiol Gastrointest Liver Physiol
272:
G1581-G1586,
1997[Abstract/Free Full Text].
19.
Inomoto, YN,
Ding M,
Nakata H,
Narumiya S,
Sugimoto Y,
Honda A,
Ichikawa A,
Chiba T,
and
Kinoshita Y.
Copresence of prostaglandin EP2 and EP3 receptors on gastric enterochromaffin-like cell carcinoid in African rodents.
Gastroenterology
109:
341-347,
1995[ISI][Medline].
20.
Jauhonen, VP,
Savolainen MJ,
and
Hassinen IE.
Cyclic AMP-linked mechanisms in ethanol-induced derangements of metabolism in rat liver and adipose tissue.
Biochem Pharmacol
24:
1879-1883,
1975[ISI][Medline].
21.
Kawada, N,
Mizoguchi Y,
Kobayashi K,
Monna T,
and
Morisawa S.
Calcium-dependent prostaglandin biosynthesis by lipopolysaccharide-stimulated rat Kupffer cells.
Prostaglandins Leukot Essent Fatty Acids
47:
209-214,
1992[ISI][Medline].
22.
Lieber, CS,
and
Savolainen M.
Ethanol and lipids.
Alcohol Clin Exp Res
8:
409-423,
1984[ISI][Medline].
23.
Nanji, AA,
Khwaja S,
Rahemtulla A,
Miao L,
Zhao S,
and
Tahan SR.
Thromboxane inhibitors attenuate pathological changes in alcoholic liver disease in the rat.
Gastroenterology
112:
200-207,
1997[ISI][Medline].
24.
Nanji, AA,
Mendenhall CL,
and
French SW.
Beef fat prevents alcoholic liver disease in the rat.
Alcohol Clin Exp Res
13:
15-19,
1989[ISI][Medline].
25.
Nanji, AA,
Miao L,
Thomas P,
Rahemtulla A,
Khwaja S,
Zhao S,
Peters D,
Tahan SR,
and
Dannenberg AJ.
Enhanced cyclooxygenase-2 gene expression in alcoholic liver disease in the rat.
Gastroenterology
112:
943-951,
1997[ISI][Medline].
26.
Nanji, AA,
Rahemtulla A,
Daly T,
Khwaja S,
Miao L,
Zhao S,
and
Tahan SR.
Cholesterol supplementation prevents necrosis and inflammation but enhances fibrosis in alcoholic liver disease in the rat.
Hepatology
26:
90-97,
1997[ISI][Medline].
27.
Pertoft, H,
and
Smedsrod B.
Separation and characterization of liver cells.
In: Cell Separation: Methods and Selected Applications, edited by Pretlow TG II,
and Pretlow TP.. New York: Academic, 1987, vol. 4, p. 1-24.
28.
Puschel, GP,
Kirchner C,
Schroder A,
and
Jungermann K.
Glycogenolytic and antiglycogenolytic prostaglandin E2 actions in rat hepatocytes are mediated via different signaling pathways.
Eur J Biochem
218:
1083-1089,
1993[Abstract].
29.
Qu, W,
Savier E,
and
Thurman RG.
Stimulation of monooxygenation and conjugation following liver transplantation in the rat: involvement of Kupffer cells.
Mol Pharmacol
41:
1149-1154,
1992[Abstract].
30.
Qu, W,
Zhong Z,
Goto M,
and
Thurman RG.
Kupffer cell prostaglandin E2 stimulates parenchymal cell O2 consumption: alcohol and cell-cell communication.
Am J Physiol Gastrointest Liver Physiol
270:
G574-G580,
1996[Abstract/Free Full Text].
31.
Regan, JW,
Bailey TJ,
Pepperl DJ,
Pierce KL,
Bogardus AM,
Donello JE,
Fairbairn CE,
Kedzie KM,
Woodward DF,
and
Gil DW.
Cloning of a novel human prostaglandin receptor with characteristics of the pharmacologically defined EP2 subtype.
Mol Pharmacol
46:
213-220,
1994[Abstract].
32.
Sato, H,
Guth PH,
and
Gossman MI.
Role of bacteria in gastric ulceration produced by indomethacin in the rat: cytoprotective action of antibiotics.
Gastroenterology
84:
483-489,
1983[ISI][Medline].
33.
Thurman, RG,
Paschal DL,
Abu-Murad C,
Pekkanen L,
Bradford BU,
Bullock KA,
and
Glassman EB.
Swift increase in alcohol metabolism (SIAM) in the mouse: Comparison of the effect of short-term ethanol treatment on ethanol elimination in four inbred strains.
J Pharmacol Exp Ther
223:
45-52,
1982[Abstract].
34.
Wendell, GD,
and
Thurman RG.
Effect of ethanol concentration on rates of ethanol elimination in normal and alcohol-treated rats in vivo.
Biochem Pharmacol
28:
273-279,
1979[ISI][Medline].
Am J Physiol Gastrointest Liver Physiol 279(1):G100-G106
0193-1857/00 $5.00
Copyright © 2000 the American Physiological Society