Stimulation of oxygen uptake by prostaglandin
E2 is oxygen dependent in perfused
rat liver
Wei
Qu,
Zhi
Zhong,
Gavin E.
Arteel, and
Ronald G.
Thurman
Laboratory of Hepatobiology and Toxicology, Department of
Pharmacology, University of North Carolina, Chapel Hill, North
Carolina 27599-7365
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ABSTRACT |
The aim of this study was to determine if the
effect of prostaglandin E2
(PGE2) on hepatic oxygen uptake
was affected by oxygen tension. Livers from fed female Sprague-Dawley
rats were perfused at normal or high flow rates (4 or 8 ml · g
1 · min
1)
to vary local oxygen tension within the liver lobule. During perfusion
at normal flow rates, PGE2 (5 µM) infusion increased oxygen uptake by about 50 µmol · g
1 · h
1;
however, when livers were perfused at high flow rates, the increase was
nearly twice as large. Simultaneously, glucose output was increased
rapidly by about 50%, whereas glycolysis was decreased about 60%.
When flow rate was held constant, increases in oxygen uptake due to
PGE2 were proportional to oxygen
delivery. Infusion of PGE2 into
livers perfused at normal flow rates increased state 3 rates of oxygen uptake of subsequently isolated
mitochondria by about 25%; however, rates were increased 50-75%
in mitochondria isolated from livers perfused at high flow rates. Thus
it is concluded that PGE2
stimulates oxygen uptake via mechanisms regulated by oxygen tension in
perfused rat liver. High flow rates also increased basal rates of
oxygen uptake: this increase was prevented by inactivation of Kupffer
cells with GdCl3. In addition,
conditioned medium from Kupffer cells incubated at high oxygen tension
(75% oxygen) stimulated oxygen uptake of isolated parenchymal cells by
>30% and elevated PGE2
production about twofold compared with Kupffer cells exposed to normal
air-saturated buffer (21% oxygen). These effects were blocked
completely by both indomethacin and nisoldipine. These data support the
hypothesis that oxygen stimulates Kupffer cells to release mediators
such as PGE2 which elevate oxygen
consumption in parenchymal cells, possibly by mechanisms involving
cyclooxygenase and calcium channels.
Kupffer cells; eicosanoids; hypermetabolic state
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INTRODUCTION |
PROSTAGLANDINS, which are metabolites of arachidonate,
are locally acting hormones that initiate a multitude of physiological actions in nearly all mammalian tissues (6, 33). They have important roles in cell-to-cell signal propagation between
nonparenchymal and parenchymal cells in liver (30). It is well known
that Kupffer cells are the major source of hepatic eicosanoids and that
hepatocytes have receptors for a variety of different classes of
eicosanoids (5, 16, 35). Eicosanoids produced by hepatic nonparenchymal cells have long been known to participate in metabolic regulation of
processes such as carbohydrate release by parenchymal cells (3).
Recently, Qu et al. (32) demonstrated that Kupffer cells were activated
by ethanol and endotoxin to release prostaglandin E2
(PGE2), which stimulated oxygen
uptake in parenchymal cells. PGE2
added directly to hepatic parenchymal cells also caused a dose-dependent increase in oxygen consumption (32); however, the
precise mechanisms by which PGE2
stimulates oxygen uptake remain unclear.
Cells in various zones of the liver lobule exist at different oxygen
tensions due to a natural oxygen gradient (17). Also, hepatocytes
located near the portal vein take up oxygen at rates two-to-three times
faster than cells located near the central vein (21). Interestingly,
hormones that increase intracellular calcium stimulate oxygen uptake
predominantly in regions of the liver lobule where oxygen tension is
the lowest (23). Oxygen also plays an important role in the regulation
of metabolism by hormones (17). For example, Kizaki and Thurman (17)
demonstrated that glucagon increased oxygen uptake of mitochondria
subsequently isolated from the perfused liver about twice as much at
high than at normal flow rates because of increased oxygen delivery.
Addition of glucagon to suspensions of mitochondria, however, had no
effect on oxygen uptake. Thus the effect of glucagon on mitochondria must be "remembered" by the organelle during the isolation
procedure (17), suggesting some permanent alteration such as
phosphorylation. In contrast, oxygen tension had little effect on
oxygen uptake in isolated hepatocytes and had virtually none in
isolated mitochondria (26). Therefore, we hypothesize that oxygen
stimulates Kupffer cells to release mediators such as
PGE2, which elevates oxygen consumption in parenchymal cells. The purpose of this study was to
determine if PGE2 stimulates
oxygen uptake in an oxygen-dependent manner in the isolated perfused
liver and if nonparenchymal cells participate in this phenomenon.
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METHODS |
Experimental animals and liver perfusion.
Female Sprague-Dawley rats (200-220 g) were allowed free access to
laboratory chow and tap water. For some experiments,
GdCl3 (10 mg/kg) dissolved in
acidified saline (pH 3.0) was injected into the tail vein 24 h before
perfusion. Details of the perfusion technique have been described
elsewhere (34). Briefly, livers were perfused with Krebs-Henseleit
bicarbonate buffer (pH 7.4, 37°C) saturated with an oxygen-carbon
dioxide mixture (95:5) in a nonrecirculating system. Perfusate was
pumped into the liver via a cannula inserted in the portal vein, and
effluent perfusate was collected via a cannula placed in the inferior
vena cava. Effluent perfusate was channeled past a Teflon-shielded,
Clark-type platinum electrode to determine oxygen tension. Rates of
oxygen uptake or metabolite production were calculated from the
difference between the influent and effluent oxygen concentration,
liver wet weight, and flow rate. Samples of effluent perfusate were collected and analyzed for glucose, lactate, and pyruvate by standard enzymatic techniques (1). Livers were perfused at normal flow rates of
~4, at medium flow rates of 6, or at high flow rates of 8 ml · g
liver
1 · min
1.
For some experiments, the perfusate oxygen concentration was varied at
constant flow rate (Table 1). At all flow
rates studied, lactate dehydrogenase, an index of cell injury, could
not be detected in the effluent perfusate, indicating that changes in
flow did not affect tissue viability.
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Table 1.
Effect of PGE2 on oxygen uptake, glycolysis, and glucose
production in livers perfused at various flow rates and different
oxygen tensions
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Isolation of mitochondria.
Mitochondria were isolated from livers perfused at normal, medium, or
high flow rates in the presence or absence of
PGE2 (5 µM) by standard
techniques of differential centrifugation. Livers were
homogenized at 0-1°C in a buffer consisting of 0.225 M
mannitol, 0.075 M sucrose, and 0.1 mM EDTA, pH 7.0, using a
Teflon-glass homogenizer. Nuclear and cellular debris were removed by
centrifugation at 2,000 g for 10 min,
and the supernatant was centrifuged subsequently at 10,000 g for 10 min. The mitochondrial pellet
was washed twice in 20 ml of buffer and was resuspended at protein
concentrations of 25-35 mg/ml (17, 31).
Measurement of mitochondrial oxygen uptake.
Mitochondrial oxygen uptake was measured at 25°C with a
Teflon-shielded, Clark-type oxygen electrode in 2 ml of a buffer (pH 7.2) containing (in mM) 100 KCl, 50 sucrose, 20 Tris · HCl, and 5 Tris phosphate, and 10 µM
rotenone (9). State 4 rates of respiration were initiated by the addition of succinate (1 µmol) and
correspond to electron flux in the absence of ADP.
State 3 rates of respiration occur
when ADP (0.5 µmol) is added and reflect near maximal rates of ATP
synthesis (4). Mitochondrial protein was determined colorimetrically
using BSA as the standard (12).
Isolation and culture of Kupffer cells.
Kupffer cells were isolated and cultured as described by Pertoft and
Smedsrod (29). Briefly, rats were anesthetized with pentobarbital (60 mg/kg ip), and the liver was isolated and perfused in a
nonrecirculating system with calcium-free Krebs-Ringer-HEPES buffer
containing 0.5 mM EGTA (pH 7.4, 37°C) for 10 min. The liver was
then perfused with Krebs-Ringer-HEPES buffer containing 0.02% type IV
collagenase (Sigma Chemical, St. Louis, MO) for 6 min. Liver cells were
dispersed by shaking gently in PBS (pH 7.4, 4°C), and the
nonparenchymal cell fraction was separated from parenchymal cells by
centrifugation through a 50% Percoll gradient (Pharmacia, Uppsala,
Sweden) (13). To purify cells and calculate the number of attached
Kupffer cells, nonparenchymal cells were resuspended in RPMI 1640 culture medium containing 15% heat-inactivated FCS, 100 U/ml
penicillin G, and 100 µg/ml streptomycin sulfate. Cells were
quantitated with a hemocytometer. About 9 × 106 nonparenchymal cells were
seeded onto each 60-mm culture dish and cultured at 37°C in a 5%
carbon dioxide atmosphere. Then 3 ml of medium containing nonadherent
endothelial and stellate cells were collected 15 min later, and 3 ml of
fresh culture medium were used to wash each dish (29). These fractions
were pooled, and the number of cells in the fraction was counted. The
number of attached Kupffer cells was calculated by subtracting the
number of cells removed from the number of cells seeded to each dish. The volume of medium was adjusted to yield 2 × 106 cells/ml. All flat cells on
the culture dish phagocytosed 1-µm latex beads, verifying that they
were Kupffer cells (9). The viability of Kupffer cells was assessed by
light microscopy and uptake of trypan blue which routinely exceeded
90%.
Measurement of parenchymal cell oxygen uptake.
Hepatocytes were isolated from rat livers according to the method of
Pertoft and Smedsrod (29). Briefly, livers were perfused with 0.02%
collagenase (Sigma) for 6-8 min until the tissue surrounding each
lobe became detached from the parenchyma. The liver was placed in cold
buffer, and hepatocytes were dispersed by gentle shaking and separated
from other cells and liver debris by centrifugation at 50 g for 2 min. Pellets were subsequently
washed with Krebs-Henseleit bicarbonate buffer and collected by
centrifugation at 50 g for 2 min (32).
Viability of hepatocytes was assessed by light microscopy and uptake of
trypan blue which routinely exceeded 90%. Kupffer cells isolated from
normal rats were cultured in 60-mm culture dishes with RPMI 1640 at
37°C in a 5% carbon dioxide atmosphere for 4 h. Subsequently,
oxygen was increased to 75% oxygen-5% carbon dioxide for 4 h. In some
experiments, indomethacin [5 µM, an inhibitor of cyclooxygenase
(COX)] or nisoldipine (4 µM, a calcium channel blocker) was
added before exposure to oxygen. Conditioned medium was collected and
incubated with parenchymal cells isolated from untreated rats in a
closed chamber (2 ml) fitted with a Clark-type oxygen electrode, and
changes in oxygen concentration were measured.
Measurement of PGE2 in conditioned
medium from cultured Kupffer cells.
Isolated Kupffer cells were cultured as previously described. Samples
of conditioned medium were analyzed for
PGE2 (20, 32) by competitive RIA
using
125I-PGE2
from Advanced Magnetics (Cambridge, MA). Although this antibody reacts
with PGE1, there is less than 2%
cross-reactivity with other prostaglandins, arachidonic acid, and
thromboxane.
Statistical analysis.
Student's t-test or ANOVA was used as
appropriate. Differences were considered significant at
P < 0.05.
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RESULTS |
Effect of PGE2 on hepatic oxygen uptake
and carbohydrate metabolism in perfused livers from fed rats.
Livers from fed rats were perfused at normal, medium, and high flow
rates to deliver oxygen to the organ at various rates. The effect of
PGE2 on oxygen uptake, glucose
output, and glycolysis (lactate plus pyruvate production) in livers
perfused at normal and high flow rates from
PGE2-treated rats is depicted in
Figs. 1 and 2
and is summarized in Table 1. At normal flow rates, basal rates of
oxygen uptake were 100-110 µmol · g
1 · h
1.
The subsequent infusion of PGE2 (5 µM) increased respiration gradually to peak values around 150 µmol · g
1 · h
1
(Fig. 1A); basal rates of glucose
output ranged from 40 to 50 µmol · g
1 · h
1,
and PGE2 increased glucose output
by about 35 µmol · g
1 · h
1
(Fig. 1B, Table 1). Concomitantly,
basal rates of production of lactate plus pyruvate (glycolysis) were 29 µmol · g
1 · h
1.
PGE2 did not influence glycolysis
significantly at normal flow rates (Fig.
1B, Table 1); however, a tendency for
a decrease was observed. In livers perfused at high flow rates (Fig.
2A, Table 1), basal rates of oxygen
uptake of 128 µmol · g
1 · h
1
were nearly doubled by PGE2. Thus
PGE2 stimulated oxygen uptake about twofold more in livers perfused at high than at normal flow rates. However, PGE2 infused at
concentrations of 10 µM in livers perfused at normal flow rates only
increased oxygen uptake from 113 to 159 µmol · g
1 · h
1;
values are similar to those observed with 5 µM
PGE2. Thus the results observed at
high flow rates cannot be explained by changes in
PGE2 delivery. Moreover, glucose
output was increased rapidly from 58 to 95 µmol · g
1 · h
1
by infusion of PGE2, whereas
glycolysis was decreased from 103 to 37 µmol · g
1 · h
1
(Fig. 2B, Table 1). When flow rate was
held constant and oxygen was varied at 50% influent oxygen
concentration, the basal rate of oxygen uptake was 102 ± 5 µmol · g
1 · h
1.
The subsequent infusion of PGE2 (5 µM) increased respiration gradually to peak values around 146 ± 4 µmol · g
1 · h
1.
However, when the perfusate was saturated with 95% oxygen, the subsequent infusion of PGE2 (5 µM) increased respiration gradually from basal levels of 128 to peak
values of 207 µmol · g
1 · h
1
(Table 1). Thus, at constant flow, the response to
PGE2 was nearly twofold greater at
95% than at 50% oxygen.

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Fig. 1.
Effect of prostaglandin E2
(PGE2) on rates of oxygen uptake,
and glucose and lactate plus pyruvate production in livers perfused at
normal flow rates. Livers from fed rats were perfused with
Krebs-Henseleit bicarbonate buffer (pH 7.4, 37°C) in a
nonrecirculating system as described in
METHODS.
PGE2 (final concentration 5 µM)
was infused with a precision infusion pump from 20 to 40 min, as
indicated by the horizontal bars and arrows. Typical experiment at
normal flow rates (4 ml · g 1 · min 1).
A: oxygen uptake by liver.
B: production of glucose and lactate
plus pyruvate. Rates were calculated from influent minus effluent
concentration differences, flow rate, and liver wet weight. Results,
with representative error terms, are from typical experiments that were
repeated 4 times in each group.
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Fig. 2.
Effect of PGE2 on rates of oxygen
uptake, and glucose and lactate plus pyruvate production in livers
perfused at high flow rates. Conditions are as described in legend for
Fig. 1 except that livers were perfused at high flow rates (8 ml · g 1 · min 1).
A: oxygen uptake by liver.
B: production of glucose and lactate
plus pyruvate. Rates were calculated from influent minus effluent
concentration differences, flow rate, and liver wet weight. Results,
with representative error terms, are from typical experiments that were
repeated 4 times in each group.
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Relationship between stimulation of oxygen uptake by
PGE2 and average hepatic oxygen
concentration.
As the oxygen concentration in the liver was increased by elevating
flow, the response of respiration to
PGE2 was increased in a flow
rate-dependent manner, reaching values around 100 µmol · g
1 · h
1.
This stimulation of oxygen uptake by
PGE2 was directly proportional to
rates of oxygen delivery when the flow rate was varied at normal flow
rates of 4, at medium flow rates of 6, or at high flow rates of 8 ml · g
liver
1 · min
1
(Fig. 3, Table 1).

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Fig. 3.
Relationship between increase in oxygen uptake by
PGE2 and average hepatic oxygen
concentration. Livers from fed rats were perfused with
PGE2 (5 µM) at normal (4 ml · g 1 · min 1),
medium (6 ml · g 1 · min 1),
and high flow rates (8 ml · g 1 · min 1)
to vary the oxygen gradient as described in
METHODS. Average hepatic oxygen
concentration equals inflow O2
plus outflow O2 divided by 2. Each
point represents data from a single liver;
r2 = 0.93. , normal flow rate;
, medium flow rate; , high flow rate; , the perfused oxygen
concentration was adjusted at 50%.
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Effect of oxygen delivery on
PGE2-stimulated mitochondrial oxygen uptake.
In mitochondria from livers perfused at normal flow rates,
state 3 rates of respiration were
elevated 25% by prior PGE2
infusion, whereas state 4 rates were
not affected (Table 2). Rates of
respiration were also increased by prior
PGE2 infusion in mitochondria
isolated from livers perfused at high flow rates; however, the effect
was much larger. For example, state 3 rates of respiration were increased about 60% by
PGE2 at high flow rates (Table 2),
whereas state 4 rates were also
increased about 40%. Thus, PGE2
stimulated oxygen uptake two- to threefold more in mitochondria
isolated from livers perfused at high than at normal flow rates.
However, addition of PGE2 to
mitochondria directly had no significant effect on either
state 3 or
4 rate of respiration. For example
state 3 rate of respiration was 16.9 ± 1.8 with PGE2 addition vs.
16.2 ± 1.2 nmol
oxygen · min
1 · mg
protein
1 in controls. State
4 values were 54.9 ± 8.5 with
PGE2 vs. 48.5 ± 3.0 nmol
oxygen· min
1 · mg
protein
1 in controls.
Inactivation of Kupffer cells prevents increases in oxygen uptake
due to PGE2 at high oxygen concentrations.
To determine if Kupffer cells are involved in stimulation of oxygen
uptake due to oxygen, livers were perfused at normal and high flow
rates to vary the oxygen gradient. In some rats,
GdCl3 (10 mg/kg) was injected into
the tail vein 24 h before perfusion to destroy Kupffer cells. As shown
in Fig. 4, basal rates of oxygen uptake
were increased about 70% by perfusion at high flow rates. GdCl3 per se did not alter basal
rates of oxygen uptake at normal flow rates; however, the increase due
to high flow rate was nearly totally prevented by
GdCl3. Furthermore, arachidonic
acid (5 µM) had no effect on oxygen uptake by isolated parenchymal
cells from normal rats (35.6 ± 0.9 vs. 37.2 ± 1.6 µmol · g
1 · h
1),
whereas PGE2 (5 µM) increased
values by nearly 50% (35.6 ± 0.9 vs. 55.4 ± 6.6 µmol · g
1 · h
1).

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Fig. 4.
Effect of destruction of Kupffer cells with
GdCl3 on oxygen uptake by perfused
livers. Livers from small rats (90-100 g) were perfused at normal
and high flow rates as described in Figs. 1 and 2. In some experiments
GdCl3 (10 mg/kg) was injected into
the tail vein 24 h before perfusion. Results are from typical
experiments that were repeated 4 times in each group. Values are means ± SE, n = 4. a P < 0.05 for
comparison with normal flow rates by ANOVA.
b P < 0.05 for
comparison with high flow rate by ANOVA.
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Stimulation of parenchymal cell oxygen uptake and
PGE2 production by conditioned medium from
Kupffer cells exposed to high oxygen tension.
Conditioned medium from Kupffer cells exposed to high oxygen tension
stimulated oxygen uptake over 30% more than medium from cells exposed
to 21% oxygen. Moreover, this effect was blocked completely by
indomethacin or nisoldipine (Fig.
5A).
Elevated oxygen tension also increased
PGE2 production about 60% by
cultured Kupffer cells, an effect that was also blocked by both
indomethacin (5 µM) and nisoldipine (4 µM; Fig.
5B). However, when indomethacin and
nisoldipine were added directly to parenchymal cells, oxygen uptake was
31.5 ± 1.5 and 31.9 ± 1.8 µl · h
1 · 106
cells
1, respectively, compared with control values of
30.3 ± 0.5 µl · h
1 · 106
cells
1. Thus indomethacin and nisoldipine do not
directly affect parenchymal cells.

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Fig. 5.
Stimulation of parenchymal cell oxygen uptake and
PGE2 production by conditioned
medium from Kupffer cells exposed to high oxygen tension. Isolated
Kupffer cells were cultured in RPMI 1640 medium with 21%
O2-5%
CO2 for 4 h, then in fresh medium
equilibrated with either 21 or 75%
O2 for an additional 4 h. A:
parenchymal cell oxygen consumption measured as described in
METHODS.
B:
PGE2 content in conditioned medium
measured as described in METHODS.
Values are means ± SE, n = 5. a P < 0.05 for
comparison with 21% O2 by ANOVA.
b P < 0.05 for
comparison with 75% O2-treated
group alone by ANOVA. 95% O2 was
toxic to cultured Kupffer cells under these conditions. Indo,
indomethacin (5 µM); Niso, nisoldipine (4 µM).
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DISCUSSION |
Stimulation of oxygen uptake by PGE2 is
dependent on oxygen tension in the liver.
Recently, Qu et al. (32) reported that Kupffer cells were activated by
ethanol and endotoxin to release mediators such as PGE2, which stimulated oxygen
consumption in parenchymal cells via mechanisms involving cell-cell
communication. From this earlier work (32) as well as this study, it is
clear that PGE2 stimulates oxygen
uptake in the liver (Fig. 1 and Table 1). Interestingly, this increase
was two to three times greater in livers perfused at high compared with
normal flow rates (Figs. 1 and 2, and Table 1) and was directly
proportional to the average oxygen concentration when the flow rate was
varied (Fig. 3). Previous work from this laboratory demonstrated that
elevation of flow rate decreased the hepatic oxygen gradient and
increased oxygen delivery to the organ (36). However, flow could modify
more than just oxygen delivery. Therefore, flow rate was held constant
and oxygen was varied. At 50% influent oxygen concentration, the basal
rate of oxygen uptake was 102 ± 5 µmol · g
1 · h
1.
The subsequent infusion of PGE2 (5 µM) increased
respiration gradually to peak values around 146 ± 4 µmol · g
1 · h
1.
However, when the perfusate was saturated with 95% oxygen, the subsequent infusion of PGE2 (5 µM) increased
respiration gradually from basal levels of 128 µmol · g
1 · h
1
to peak values of 207 µmol · g
1 · h
1
(Table 1). At constant flow, the response to PGE2 was
nearly twofold greater than at 50% oxygen. Thus it is clear that
increases in oxygen delivery to the liver than changes in flow rate are responsible for changes in respiration observed in this study. Moreover, PGE2 increased oxygen uptake about twice as
much in subsequently isolated mitochondria at high than at normal flow rates (Table 2). Thus the oxygen-dependent action of
PGE2 involves mitochondria and is a "remembered"
event (i.e., it is not lost during the isolation
procedure).
The question then arises, how does
PGE2 increase oxygen uptake in the
liver? About 25% of the increase can be accounted for by enhanced
demand of mitochondrial oxidative phosphorylation for oxygen to
compensate for reduced extramitochondrial ATP production due to
inhibition of glycolysis (17). For example, at high flow rates, oxygen
uptake was increased from 128 to 207 µmol · g
1 · h
1,
whereas glycolysis was reduced from 103 to 37 µmol · g
1 · h
1.
It is known that glycolysis from glycogen yields a net 1.5 mol ATP/mol
lactate plus pyruvate produced (17). Thus the decrease of glycolysis is
equivalent to 1.5 × (103
37) = 99 µmol
ATP · g
1 · h
1.
In contrast, consumption of 79 µmol
oxygen · g
1 · h
1
by the mitochondrial respiratory chain produces 426 µmol
ATP · g
1 · h
1.
Thus decreased glycolytic ATP production can only account for about
25% (99 of 426) of the increase in oxygen uptake due to PGE2. However, factors responsible
for the predominant fraction (i.e., 75%) of the increase in oxygen
uptake due to PGE2 remain unclear.
DeRubertis et al. (7) demonstrated that high oxygen tension (95%
oxygen-5% carbon dioxide) stimulated cAMP production about 6- to
10-fold in the inner medulla of the kidney and that the cAMP regulatory
system was sensitive to tissue oxygen tension (24). The
PGE2-induced increase in oxygen
uptake involves EP2 receptors, G
proteins, the cAMP signal transduction pathway, protein kinase A, and
mitochondria (W. Qu, L. M. Graves, and R. G. Thurman, unpublished
data). Thus enhanced cAMP production may explain the rest
of the increase in oxygen uptake caused by
PGE2.
Involvement of Kupffer cells in mechanisms of increased oxygen
uptake.
It has been reported that cultured nonparenchymal cells produce a
variety of eicosanoids from arachidonate (27, 28). Eicosanoids produced
by hepatic nonparenchymal cells such as
PGE2 participate in metabolic
regulation of processes such as carbohydrate release by parenchymal
cells (2). Recently, Qu et al. (32) demonstrated that oxygen uptake of
parenchymal cells from normal rats was stimulated 30-40% by
conditioned medium collected from Kupffer cells isolated from
ethanol-treated rats. Therefore, intercellular communication in the
liver is a potentially important mechanism for the regulation of
hepatic metabolism (10, 14, 18, 19). In adult rats, the effect of
oxygen on basal rates of oxygen uptake was small (Table 1). Thus oxygen
may increase the sensitivity of parenchymal cells to
PGE2. However, basal rates of
oxygen uptake were nearly doubled after perfusion at high flow rates in
livers from small rats (Fig. 4). Elevated oxygen uptake due to oxygen
was blocked by treatment with
GdCl3, supporting the hypothesis
that Kupffer cells are involved. In addition,
PGE2 added directly to isolated parenchymal cells increased oxygen uptake (32), supporting the hypothesis that Kupffer cells release eicosanoids in response to
oxygen, which regulates oxygen metabolism in parenchymal cells.
Oxygen stimulates PGE2 production by
Kupffer cells, which increases oxygen uptake in hepatic parenchymal
cells.
Previous work from this laboratory showed that basal rates of oxygen
uptake were about two times higher in periportal than in pericentral
regions of the liver lobule when liver perfusion was in the anterograde
direction. When the direction of perfusion was reversed, oxygen uptake
was nearly three times higher in pericentral than in periportal
regions. Thus rates of oxygen uptake were higher in "upstream"
than in "downstream" regions of the liver lobule regardless of
the direction of the flow (21, 22). These results support the
hypothesis that oxygen tension regulates oxygen uptake in the liver.
Later, Nakagawa et al. (26) observed that an increase in oxygen uptake
due to arachidonate, which elevates intracellular calcium and is
metabolized predominantly in Kupffer cells to eicosanoids, was two- to
threefold greater at high than at low initial oxygen tensions (26).
Furthermore, arachidonate increased oxygen uptake to a much greater
extent in downstream than in upstream regions of the liver lobule.
Collectively, these results suggest that the endogenous regulator(s) of
oxygen metabolism in hepatic parenchymal cells is produced from
arachidonate by Kupffer cells. In this study, it was demonstrated that
conditioned medium collected from Kupffer cells exposed to high oxygen
produced PGE2 and stimulated respiration in isolated parenchymal cells (Fig. 5). Therefore, the
rapid stimulation in oxygen uptake by ethanol treatment and oxygen may
have common pathways involving Kupffer cells. These data clearly
support the hypothesis that Kupffer cells participate indirectly in the
mechanism of elevated oxygen metabolism in hepatic parenchymal cells by
producing mediators that stimulate parenchymal cell oxygen metabolism.
Because oxygen tension has little direct effect on oxygen uptake in
isolated hepatocytes and no effect in isolated mitochondria other than
to saturate cytochrome oxidase (25), whereas arachidonic acid
stimulated oxygen uptake in perfused liver but not in isolated hepatocytes (26), it is proposed that an oxygen sensor exists in
Kupffer cells that produces mediators such as
PGE2 in response to oxygen, which
stimulates oxygen uptake in parenchymal cells (Fig.
6). An interesting question arising from
this work is what is the nature of this proposed oxygen sensor in
Kupffer cells? Because the Michaelis constant of COX for oxygen is only
20 µM (21), it is an unlikely oxygen sensor. In contrast, COX is the rate-limiting enzyme in prostanoid synthesis, and oxidant stress is an
inducer of COX-2 gene expression (11). This study showed that
indomethacin, a nonspecific COX inhibitor, prevented stimulation of
oxygen uptake due to conditioned medium from Kupffer cells exposed to
high oxygen tension (Fig. 5). Thus COX-2 could be involved in an oxygen
sensor mechanism. Furthermore, calcium is necessary for phospholipase
A2 activation and eicosanoid
synthesis (8). Nisoldipine, a calcium channel blocker, prevented
stimulation of oxygen uptake due to conditioned medium from Kupffer
cells exposed to high oxygen tension (Fig. 5), suggesting that
intracellular calcium could also be involved in an oxygen sensor
mechanism. The determination of the precise pathways involved in a
proposed oxygen sensor mechanism in Kupffer cells remains an important gap in our knowledge.

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Fig. 6.
Scheme depicting working hypothesis for how oxygen and
PGE2 increase hepatic oxygen
consumption. Oxygen increases intracellular calcium in Kupffer cells,
which in turn activates phospholipase
A2 and increases the rate of
PGE2 synthesis most likely via
mechanisms involving COX-2. PGE2
then acts on receptors in parenchymal cells to stimulate mitochondrial
respiration via pathways involving cAMP. COX-2, cyclooxygenase-2; AA,
arachidonic acid.
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In conclusion, high oxygen tension stimulates Kupffer cells to release
mediators such as PGE2, which
increases oxygen consumption in parenchymal cells. Furthermore,
PGE2 also stimulates oxygen uptake
in parenchymal cells via oxygen-dependent pathways involving cAMP (Fig.
6).
 |
ACKNOWLEDGEMENTS |
We thank the Center for Gastrointestinal Biology and Disease
(supported by the National Institute of Diabetes and Digestive and
Kidney Diseases Grant P30-DK-34987) for assistance with the PGE2 measurements. The study was
supported in part by the National Institute of Alcohol Abuse and
Alcoholism Grants AA-09156 and AA-03624. W. Qu was also supported
partially by an award from the Institute of Nutrition, Univ. of North
Carolina.
 |
FOOTNOTES |
Address for reprint requests: R. G. Thurman, Laboratory of
Hepatobiology and Toxicology, Dept. of Pharmacology, CB 7365, Faculty
Laboratory Office Building, Univ. of North Carolina, Chapel Hill, NC
27599-7365.
Received 23 November 1996; accepted in final form 12 May 1998.
 |
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