PGE2 stimulates
O2 uptake in hepatic parenchymal
cells: involvement of the cAMP-dependent protein
kinase
Wei
Qu,
Lee M.
Graves, and
Ronald G.
Thurman
Laboratory of Hepatobiology and Toxicology, Department of
Pharmacology, University of North Carolina at Chapel Hill, Chapel
Hill, North Carolina 27599-7365
 |
ABSTRACT |
The aim of this study was to determine which
PGE2 receptors and signal
transduction pathways are responsible for the stimulation of oxygen
uptake in liver. Hepatic parenchymal cells isolated from female
Sprague-Dawley rats were incubated either with
PGE2, 17-phenyl-omega-trinor
PGE2 (an EP1-specific
agonist), or 11-deoxy PGE1 (an
EP2/EP4-specific agonist), and oxygen
consumption was measured. Both
PGE2 and 11-deoxy
PGE1 stimulated oxygen
consumption. However, an EP1
agonist was without effect. Although
PGE2 elevated intracellular
calcium, this occurred at concentrations ~500-fold lower than that
required to stimulate oxygen uptake.
PGE2-stimulated increases in cAMP
formation correlated well with the increase in oxygen consumption.
Dibutyryl cAMP also increased oxygen consumption. Furthermore,
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, a cell-permeable inhibitor of protein kinase A (PKA), reduced the
stimulation of oxygen uptake by
PGE2. Incubation of isolated parenchymal cell mitochondria with the purified catalytic subunit of
PKA and ATP increased both state 3 rates of oxygen uptake and the
respiratory control ratio by ~50%. Activation of these events was
prevented by incubation with the PKA inhibitory peptide, PKI. These
findings are consistent with the hypothesis that
PGE2 stimulates oxygen consumption
via an EP2 and/or
EP4 subclass of receptors through
the actions of cAMP on a cAMP-dependent protein kinase.
prostaglandin E2; protein
kinase A; mitochondria
 |
INTRODUCTION |
PROSTAGLANDINS ARE locally acting hormones that have a
remarkable variety of physiological actions in nearly all mammalian tissues. Prostaglandins are key mediators of cell signaling between nonparenchymal and parenchymal cells in the liver (19),
and Kupffer cells are the main sources of prostanoids in this tissue (1). For example, Casteleijn et al. (2)
demonstrated that prostaglandins from Kupffer cells stimulated
glycogenolysis in liver parenchymal cells. Recently, it has been
demonstrated that ethanol and endotoxin stimulated Kupffer cells to
release PGE2, which in turn
increased oxygen uptake in parenchymal cells (21). Many of the known
biological effects of PGE2 are
mediated through interaction of
PGE2 with specific receptors (5,
8). Until now, four different genes coding for
PGE2 receptors (EP receptors) have
been cloned, and four subtypes of receptors
(EP1,
EP2,
EP3, and
EP4) have been characterized
pharmacologically on the basis of the relative agonist or antagonist
potencies of a number of different analogs of PGE2 from at
least one species (5, 15). 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 calcium concentration
([Ca2+]i).
EP2 and
EP4 receptors act via
Gs proteins to mediate an increase in cAMP, whereas EP3 receptors are
coupled to Gi and decrease cAMP
(19).
Phosphorylation by the cAMP-dependent protein kinase A (PKA) plays a
pivotal role in the transduction of signals by hormones such as
prostaglandins in eukaryotic cells (27). To better understand the
mechanism by which oxygen uptake is regulated, the subtypes of
PGE2 receptors and signaling
pathways were investigated. The results of this study are consistent
with the hypothesis that PGE2 acts
through the EP2 and/or
EP4 receptor subtype to increase cAMP, which activates cAMP-dependent PKA, leading to increased oxygen
uptake in hepatic parenchymal cells.
 |
EXPERIMENTAL PROCEDURES |
Experimental animals and materials.
Female Sprague-Dawley rats (200-240 g) were
allowed free access to laboratory chow and tap water. Fed animals were
used in all experiments. All animals were given humane care in
compliance with institutional guidelines. 11-Deoxy PGE1 and
17-phenyl-omega-trinor PGE2 were
obtained from Cayman Chemical.
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) dihydrochloride was obtained from Calbiochem, and type IV
collagenase was purchased from Sigma (St. Louis, MO). PKA was obtained
from New England Biolabs. Kemptide, the PKI peptide, and all other
chemicals were obtained from Sigma.
Isolation and culture of parenchymal cells.
Parenchymal cells were isolated from rat liver
according to the method of Pertoft and Smedsrod (18). Briefly, livers
were isolated under pentobarbital anesthesia (60 mg/kg ip) 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 (pH 7.4, 115 mM NaCl,
5 mM KCl, 1 mM
KH2PO4,
25 mM HEPES, and 1 mM CaCl2)
containing 0.02% type IV collagenase at 37°C for 6-8 min
until the tissue surrounding the lobes became detached from the
parenchyma. The liver was placed in cold buffer, and parenchymal cells
were dispersed by gentle shaking and separated from other cells and
liver debris by centrifugation at 50 g
for 2 min. Cells were subsequently washed with Krebs-Henseleit
bicarbonate buffer and collected by centrifugation at 50 g for 2 min as described previously
(21). Additional washing was performed to remove residual
nonparenchymal liver cells. Cultured hepatocytes were free of
contamination by other nonparenchymal liver cells, such as Kupffer and
stellate cells, as assessed microscopically. Viability of parenchymal
cells was assessed by light microscopy and uptake of trypan blue (13)
and routinely exceeded 90%. Isolated parenchymal cells were
resuspended in DMEM-F-12 culture medium, seeded onto 25-mm
glass coverslips in 60-mm culture dishes, and cultured at 37°C in a
5% CO2 atmosphere. Cultured
parenchymal cells were used for this study within 8 h of isolation.
Isolation of mitochondria.
Mitochondria were isolated from rat liver by standard
techniques of differential centrifugation (25). 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 (14).
Measurement of oxygen uptake.
Isolated parenchymal cells were incubated with RPMI
1640 culture medium at 37°C in a closed chamber (2 ml) fitted with
a Clark-type oxygen electrode, and oxygen consumption was measured as
described elsewhere (21). In some experiments, isolated parenchymal
cells were incubated with either antimycin A (10 µM) or
potassium cyanide (2 mM), and then oxygen consumption was measured with
addition of PGE2 as described
above. Mitochondrial oxygen uptake was measured at
25°C with a Teflon-shielded, Clark-type oxygen electrode in 2 ml of
a buffer containing 20 mM Tris (pH 7.2), 100 mM KCl, 50 mM sucrose, 5 mM Tris phosphate, and 10 µM rotenone (14). Respiration was initiated
by the addition of succinate (1 µmol) and ADP (0.5 µmol).
In some experiments, isolated mitochondria (0.3-0.4 mg protein)
were incubated with 2 µg/ml of the purified catalytic subunit of PKA
in kinase buffer containing 20 mM HEPES, pH 7.3, 10 mM
-glycerophosphate, 1.5 mM EGTA, 0.1 mM
Na3VO4,
1 mM dithiothreitol, 10 mM MgCl2,
and 100 µM ATP. Isolated mitochondria were also incubated with PKA
plus the PKA inhibitory peptide PKI (10 µM). After incubation on ice
for 20 min, mitochondrial respiration was measured as described above. The amount of mitochondrial protein used in these
experiments was determined colorimetrically by employing BSA as the
standard (9).
Measurement of
[Ca2+]i.
Free [Ca2+]i in individual parenchymal cells
was assessed fluorometrically using the calcium indicator fura 2 and a
microspectrofluorometer (23). Parenchymal cells were incubated in
DMEM-F-12 culture medium containing 5 µM fura 2-AM (Molecular Probes,
Eugene, OR) and 0.06% Pluronic F-127 (BASF Wyandotte, Wyandotte, MI)
at 37°C for 30-40 min. Coverslips plated with parenchymal
cells were rinsed and placed in a chamber with Krebs-Ringer-HEPES
buffer containing 1 mM MgSO4 and 5 mM glucose at 25°C. Changes in fluorescence intensity of fura 2 at
excitation wavelengths of 340 nm and 380 nm were monitored in
individual cells with a PTI fluorescence analytical system (Photon
Technology International, South Brunswick, NJ) interfaced with a Nikon
Diaphot inverted microscope (26). Each value was corrected by
subtracting the system dark noise and autofluorescence, assessed by
quenching fura 2 fluorescence with
Mn2+.
[Ca2+]i
was calculated as described by Grynkiewicz et al. (12) and Ratto et al.
(23) from the equation
|
|
where
Fo/Fs
is the ratio of fluorescent intensities evoked by 380 nm light from
fura 2 pentapotassium salt in buffered salt solutions containing
nanomolar calcium and millimolar calcium, R is the ratio of fluorescent
intensities at excitation wavelengths of 340 nm and 380 nm, and
Rmin and
Rmax are values of R at nanomolar calcium and millimolar calcium, respectively. The values of these constants were determined at the end of each experiment, and a dissociation constant
(Kd) value of
135 nmol/l was used (12).
In all experiments, parenchymal cells were incubated in
Krebs-Ringer-HEPES buffer, and basal
[Ca2+]i was determined in the
presence of 5 mmol/l
K+.
Measurement of cAMP.
Intracellular cAMP was measured in suspensions of
parenchymal cells by radioimmunoassay using
125I-labeled-cAMP from Biomedical
Technologies (24). 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 visible pellets were separated from supernatant. Radioactivity in the precipitate was counted and compared with known values from a
standard curve.
Statistical analysis.
Student's t-test and
ANOVA were used as appropriate. Differences were considered significant
at P < 0.05.
 |
RESULTS |
Effects of PGE2 receptor subtype-specific
agonists on oxygen consumption in isolated hepatic parenchymal cells.
Isolated parenchymal cells were incubated with
PGE2 (5 µM; an agonist for all
PGE2 receptors),
17-phenyl-omega-trinor PGE2 (0.1 µM; an EP1-specific agonist), or
11-deoxy PGE1 (0.1 µM, an EP2/EP4-specific
agonist), and oxygen consumption was measured in a closed chamber with
an oxygen electrode. Treating cells with PGE2 increased oxygen consumption
by nearly 50%, and the
EP2/EP4 subtype-specific agonist 11-deoxy
PGE1 elevated oxygen uptake by
~35% (Figs. 1 and
2).
PGE2 added directly to parenchymal
cells caused a dose-dependent increase in oxygen consumption that
was linear in the concentration range from 0.005 to
0.1 µM (Fig. 3). In contrast, the
EP1 subtype-specific agonist
17-phenyl-omega-trinor PGE2 did
not affect oxygen consumption at concentrations ranging from
0.005 to 10 µM.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 1.
Effects of PGE2 and structural
analogs on oxygen consumption by isolated hepatic parenchymal cells.
PGE2 (5 µM),
17-phenyl-omega-trinor PGE2
(17-PGE2, 0.1 µM), and 11-deoxy
PGE1
(11-PGE1, 0.1 µM) were added to
isolated parenchymal cells as indicated, and changes in oxygen
concentration were measured as described in
EXPERIMENTAL PROCEDURES (typical
experiments). Rates of oxygen uptake in parentheses (microliters per
hour per 106 cells) were
calculated from the change in oxygen concentration per unit
time.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of PGE2 receptor
subtype-specific agonists on oxygen consumption in isolated hepatic
parenchymal cells.
PGE2 (5 µM),
17-PGE2 (0.1 µM), and
11-PGE1 (0.1 µM) were added to
isolated parenchymal cells, and changes in oxygen concentration were
measured as described in EXPERIMENTAL
PROCEDURES. Values are means ± SE
(n = 4-5).
* P < 0.05 for comparison
between basal, PGE2,
17-PGE2, and
11-PGE1 treatment groups by
ANOVA.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3.
11-PGE1 stimulates oxygen
consumption of parenchymal cells in a dose-dependent manner.
Parenchymal cells were isolated from normal rats and incubated in RPMI
1640 culture medium in a closed chamber.
11-PGE1 was added at
concentrations indicated on the abscissa, and oxygen consumption was
measured as described in EXPERIMENTAL
PROCEDURES. Values are means ± SE
(n = 4-5).
|
|
Because PGE2 increases
[Ca2+]i
in other cell types (7), the ability of
PGE2 to stimulate
[Ca2+]i
and oxygen uptake in isolated parenchymal cells was examined. Isolated
parenchymal cells were loaded with fura 2, and the amount of
[Ca2+]i
was determined as described in EXPERIMENTAL
PROCEDURES. Adding PGE2 increased
[Ca2+]i
in a dose-dependent manner, with the half-maximal effect occurring at
0.8 nM PGE2. However, at this
concentration PGE2 did not alter oxygen consumption (21), supporting the hypothesis that the PGE2-stimulated increase in
[Ca2+]i
was not sufficient to account for the effects of
PGE2 on oxygen consumption in
parenchymal cells observed here.
PGE2 increases intracellular cAMP in
isolated parenchymal cells in a dose-dependent manner.
PGE2 is known to
stimulate cAMP formation in some cell types through a
Gs-dependent activation of
adenylate cyclase (11). To determine if cAMP was involved in the
mechanism of PGE2-elevated oxygen
uptake, dose-response curves for
PGE2-stimulated cAMP generation and oxygen uptake were compared.
PGE2 increased cAMP formation in a
dose-dependent manner. The peak of cAMP formation occurred within 5 min
of PGE2 addition and was enhanced
by incubation of isolated parenchymal cells with IBMX (0.5 mM), a
cell-permeable phosphodiesterase inhibitor (Fig.
4). Similarly, IBMX enhanced the
PGE2-stimulated increase in oxygen
uptake from 45 ± 2 to 51 ± 7 µl · h
1 · 106
cells
1, and cAMP formation
correlated well with oxygen uptake (Fig. 5,
r2 = 0.96). Comparing the
PGE2-dependent stimulation of cAMP
formation and oxygen uptake demonstrated that the half-maximal effect
(1 µM) for these two events was identical (Fig. 4 and see also Fig. 6 in Ref. 30).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
PGE2 increases intracellular cAMP
in isolated parenchymal cells in a dose-dependent
manner. PGE2 was
added at concentrations indicated on the abscissa, and intracellular
cAMP in suspensions of isolated rat parenchymal cells was measured as
described in EXPERIMENTAL PROCEDURES.
Some cells were pretreated with IBMX (0.5 mM), a
cAMP-phosphodiesterase inhibitor, before the addition of
PGE2. , IBMX-treated cells;
, cells without IBMX. IBMX alone had no effect on either cAMP
formation or oxygen consumption. Values are means ± SE
(n = 5).
|
|

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 5.
Relationship between stimulation of oxygen consumption and cAMP
production by PGE2 in isolated
parenchymal cells. Increases in oxygen consumption by
PGE2 (5 µM) in isolated
parenchymal cells from individual livers and cAMP production in
suspensions of isolated parenchymal cells treated with
PGE2 (5 µM) were measured at the
same time and were plotted. Each point represents data from a single
liver; r2 = 0.96.
|
|
To further substantiate a role for cAMP in the response to
PGE2, cell-permeable cAMP analogs
were examined for their ability to stimulate oxygen uptake. Dibutyryl
cAMP and 8-bromoadenosine cAMP were added directly to isolated
parenchymal cells, and oxygen uptake was measured as described in
EXPERIMENTAL PROCEDURES. Both dibutyryl cAMP
and 8-bromoadenosine cAMP (1-25 µM) increased oxygen consumption
by parenchymal cells in a dose-dependent manner (Fig. 6 and data not shown).

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 6.
Dibutyryl cAMP stimulates oxygen consumption of parenchymal cells in a
dose-dependent manner. Parenchymal cells were isolated
from normal rats and incubated with RPMI 1640 culture medium in a
closed chamber fitted with a Clark-type oxygen electrode. Dibutyryl
cAMP was added at concentrations indicated on the abscissa, and oxygen
consumption was measured as described in EXPERIMENTAL
PROCEDURES. Values are means ± SE
(n = 4).
|
|
An inhibitor of PKA prevents
PGE2-stimulated oxygen uptake.
Since these results suggested that the stimulation of
oxygen consumption by PGE2 was
cAMP dependent, the requirement for PKA was examined by using a
cell-permeable inhibitor of this kinase, H-89 (4). Isolated parenchymal
cells were treated with H-89 (20 µM) before the addition of
PGE2, and oxygen consumption was measured. Incubation with H-89 reduced the stimulation of oxygen uptake
slightly but significantly from 45 ± 2 to 31 ± 3 µl · h
1 · 106
cells
1
(P < 0.05), consistent with the
hypothesis that PKA is required for stimulation of oxygen consumption
by PGE2.
PGE2-stimulated oxygen uptake requires an
intact mitochondrial respiratory chain.
Intracellular oxygen consumption occurs primarily in
the mitochondria and requires an intact mitochondrial respiratory chain (20). Antimycin A and potassium cyanide are inhibitors of the respiratory chain, and these compounds inhibit electron transport between cytochrome b and cytochrome
c and between mitochondrial cytochrome
oxidase and oxygen, respectively. To determine if an intact
mitochondrial respiratory chain was required for the effect of
PGE2, isolated parenchymal cells
were incubated with either antimycin A (10 µM) or potassium cyanide
(2 mM), and oxygen consumption was measured. Antimycin A reduced the
PGE2-stimulated increase in oxygen
uptake from 45 ± 2 to 11 ± 2 µl · h
1 · 106
cells
1
(P < 0.05), whereas potassium
cyanide totally blocked oxygen consumption in isolated parenchymal
cells. These results demonstrate that a functional mitochondrial
respiratory chain was required for this process.
The catalytic subunit of PKA stimulates mitochondrial
respiration.
These results suggested that the effect of
PGE2 on oxygen uptake was
dependent on both cAMP formation and an intact mitochondrial respiratory chain. To further examine the involvement of cAMP in this
process, the ability of PKA to directly influence mitochondrial oxygen
consumption was investigated in vitro. Intact mitochondria were
isolated by differential centrifugation and incubated with the
catalytic subunit of PKA and
Mg2+/ATP as described in
EXPERIMENTAL PROCEDURES. Oxygen uptake
in mitochondria was measured with a Clark-type oxygen electrode after addition of succinate (1 µmol; state 4) and ADP (0.5 µmol; state 3). Respiratory ratios were calculated from the state 3-to-state 4 ratio. As shown in Fig. 7 and Table
1, incubation with PKA increased both state
3 rates of oxygen uptake and the respiratory control ratio by ~50%.
The effect of PKA on mitochondrial respiration required both the
catalytic subunit of PKA and Mg2+-ATP. Neither
Mg2+-ATP nor PKA alone increased the rate of mitochondrial
respiration. The effect of PKA on mitochondrial respiration was
inhibited by coincubation with the specific PKA inhibitor peptide PKI
(Fig. 7, Table 1, and Ref. 10). As expected, PKI inhibited the
PKA-dependent phosphorylation of Kemptide, a peptide substrate for PKA
(PKA = 41.0 pmol · min
1 · ml
1;
PKA + PKI = 8.4 pmol · min
1 · ml
1;
Refs. 10, 17, and 29). Furthermore, addition of ADP during the middle
of state 3 respiration did not further stimulate oxygen uptake,
indicating that the effect of PKA on mitochondrial respiration was
direct and not due to formation of ADP as a product of the kinase
reaction.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of protein kinase A (PKA) on oxygen uptake by isolated
mitochondria. Mitochondria were isolated from rat liver
and incubated at room temperature in a 2-ml volume as described in
EXPERIMENTAL PROCEDURES. Oxygen
concentration was measured in a closed vessel with a Clark-type oxygen
electrode after addition of succinate (1 µmol) and ADP (0.5 µmol)
in control, PKA (2 µg/ml), and PKA inhibitor (PKI; 10 µM) + PKA-treated mitochondria (typical experiments). Rates of oxygen uptake
in parentheses (nanomoles per minute per milligram of protein) were
calculated from the change in oxygen concentration per unit time.
|
|
 |
DISCUSSION |
PGE2 stimulates oxygen uptake in
parenchymal cells via EP2 receptors.
Recently, Qu et al.
(21) demonstrated that alcohol and endotoxin stimulated hepatic Kupffer
cells to release PGE2, which stimulated oxygen uptake in isolated parenchymal cells; in other studies, PGE2 increased oxygen
uptake in perfused liver (22). One of the objectives of this study was
to determine the PGE2 receptor
subtype that was responsible for regulating oxygen uptake in
parenchymal cells. On the basis of the findings that the
EP2/EP4-specific agonist 11-deoxy PGE1 stimulated
oxygen consumption, whereas the EP1-specific agonist
17-phenyl-omega-trinor PGE2 was
without effect, it is concluded that in hepatic parenchymal cells
PGE2 stimulates oxygen consumption
via the EP2 and/or EP4 subclass of receptors.
cAMP but not calcium is involved in the stimulation of oxygen uptake
by PGE2.
Calcium stimulates oxygen uptake in isolated mitochondria (3, 16);
however, stimulation by cAMP-linked agonists can occur in the absence
of calcium (6). To establish the mechanism by which
PGE2 signals in parenchymal cells,
the relative contribution of calcium and cAMP to the increase in oxygen
consumption was compared. The concentration of
PGE2 that was required to
stimulate oxygen consumption was ~500-fold higher than that necessary
to increase calcium. Although these studies do not exclude a role for
calcium in the stimulation of oxygen uptake, it is unlikely that the
EP1-mediated elevation of
cytosolic calcium per se can explain
the stimulation of oxygen consumption by
PGE2. In addition, the calcium-
and phospholipid-activated protein kinase C (PKC) is also not likely to
be a mediator of PGE2-stimulated
oxygen consumption because addition of phorbol 12-myristate 13-acetate, an activator of PKC, did not affect oxygen consumption in parenchymal cells (data not shown).
cAMP-dependent protein kinase regulates mitochondrial
respiration.
The results presented here are consistent with a requirement for cAMP
and PKA in the PGE2-stimulated
increase in oxygen consumption. PGE2 has been known to increase
cAMP formation and PKA activity in human and animal cells (10, 11, 28).
In this study, PGE2 resulted in a
dose-dependent increase in cAMP in isolated parenchymal cells (Fig. 4).
The effect of PGE2 on cAMP
formation and oxygen consumption correlated well, and a comparison of
the half-maximal (1 µM) effects of
PGE2 on these processes
demonstrated remarkable similarity. Moreover, addition of cAMP analogs
produced a dose-dependent increase in oxygen consumption that mimicked
the effect of PGE2 in isolated
parenchymal cells (Fig. 6). Therefore, these observations strongly
support the hypothesis that the cAMP pathway is pivotal in the
stimulation of oxygen consumption by
PGE2.
Additional evidence suggests the direct involvement of PKA in the
regulation of respiration by PGE2.
The PGE2-induced increase in
oxygen uptake was inhibited by H-89, a PKA inhibitor. Furthermore, incubating isolated mitochondria with the purified catalytic subunit of
PKA increased both state 3 rates of oxygen uptake and the respiratory control ratio in isolated mitochondria, an effect that could be prevented by a specific peptide inhibitor of PKA, PKI (Table 1). Thus
it is proposed that PGE2
stimulates oxygen uptake by activation of PKA, leading to direct
phosphorylation of mitochondrial components that ultimately regulate
mitochondrial oxygen uptake. Future work will be necessary to identify
the PKA substrates that may be responsible for regulation of this event.
In summary, the results reported here are consistent with the
hypothesis that PGE2 activates PKA
via an EP2 and/or
EP4 receptor-adenylyl cyclase
coupled response, resulting in the phosphorylation and activation of a
protein or proteins in the mitochondrial membrane leading to increased
electron flux and respiration (Fig.
8). The mechanism by which cAMP and PKA
regulate oxygen respiration remains to be resolved. Whether or not PKA
directly phosphorylates components involved in respiratory control or
indirectly influences respiration (e.g., by regulating mitochondrial
swelling) remains to be elucidated. However,
PGE2 indeed causes mitochondrial
swelling (Qu et al., unpublished data), which stimulates mitochondrial
respiration. Furthermore, the effect of
PGE2 on respiration was blocked by an uncoupler, 2,4-dinitrophenol. Thus
PGE2 stimulates coupled oxidative
phosphorylation via the mechanism described above.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 8.
Diagram depicting working hypothesis.
PGE2 interacts with
EP2/EP4
receptors, which are coupled to adenylate cyclase (AC), leading to
increases in cAMP in hepatic parenchymal cells. cAMP activates the
cAMP-dependent PKA (the active protein kinase), which most likely
phosphorylates key mitochondrial proteins and stimulates mitochondrial
oxygen uptake. G, G protein; PKI, inactive protein kinase; PP,
phosphoprotein.
|
|
 |
ACKNOWLEDGEMENTS |
We appreciate the assistance of the Center for Gastrointestinal
Biology and Disease (supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant P30 DK-34987) with the cAMP measurements.
 |
FOOTNOTES |
This work was supported, in part, by National Institute on Alcohol
Abuse and Alcoholism Grants AA-09156 and AA-03624 (R. G. Thurman). L. M. Graves was supported by grants from the
National Institutes of Health (GM-54010) and the American Heart
Association (North Carolina Affiliate). W. Qu was also supported
partially by an award from the Institute of Nutrition, University of
North Carolina.
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, Faculty Laboratory, Office Bldg., Univ. of North Carolina at
Chapel Hill, Chapel Hill, NC 27599-7365 (E-mail:
thurman{at}med.unc.edu).
Received 8 December 1998; accepted in final form 5 August 1999.
 |
REFERENCES |
1.
Altin, J. G.,
and
F. L. Bygrave.
Non-parenchymal cells as mediators of physiological responses in liver.
Mol. Cell. Biochem.
83:
3-14,
1988[Medline].
2.
Casteleijn, E.,
J. Kuiper,
H. C. J. Van Rooij,
J. A. A. M. Kamps,
J. F. Koster,
and
T. J. C. Van Berkel.
Prostaglandin D2 mediates the stimulation of glycogenolysis in the liver by phorbol ester.
Biochem. J.
250:
77-80,
1988[Medline].
3.
Chance, B.
The energy-linked reaction of calcium with mitochondria.
J. Biol. Chem.
240:
2729-2748,
1965[Free Full Text].
4.
Chijiwa, T.,
A. Mishima,
M. Hagiwara,
M. Sano,
K. Hayashi,
T. Inoue,
K. Naito,
T. Toshioka,
and
H. Hidaka.
Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly snythesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells.
J. Biol. Chem.
265:
5267-5272,
1990[Abstract/Free Full Text].
5.
Coleman, R. A.,
I. Kennedy,
P. P. A. Humphrey,
K. Bunce,
and
P. Lumley.
Prostanoids and their receptors.
In: Comprehensive Medicinal Chemistry, edited by C. Hansch,
P. G. Sammes,
J. B. Taylor,
and J. C. Emmett. Oxford, UK: Pergamon, 1989, p. 643-714.
6.
Crompton, M.,
and
T. P. Goldstone.
The involvement of calcium in the stimulation of respiration in isolated rat hepatocytes by adrenergic agonists and glucagon.
FEBS Lett.
204:
198-202,
1986[Medline].
7.
Fang, M. A.,
D. A. Kujubu,
and
T. J. Hahn.
The effects of prostaglandin E2, parathyroid hormone, and epidermal growth factor on mitogenesis, signaling, and primary response genes in UMR 106-01 osteoblast-like cells.
Endocrinology
131:
2113-2119,
1992[Abstract].
8.
Funk, C.,
L. Furci,
G. A. Fitzgerald,
R. Grygorczyk,
C. Rochette,
M. A. Bayne,
M. Abramovitz,
M. Adam,
and
M. K. Metters.
Cloning and expression of a cDNA for human prostaglandin E receptor EP1 subtype.
J. Biol. Chem.
268:
26767-26772,
1993[Abstract/Free Full Text].
9.
Gornall, A. G.,
C. J. Bardawill,
and
M. M. David.
Determination of serum proteins by means of the biuret reaction.
J. Biol. Chem.
177:
751-766,
1949[Free Full Text].
10.
Graves, L. M.,
K. E. Bornfeldt,
E. W. Raines,
B. C. Potts,
S. G. Macdonald,
R. Ross,
and
E. G. Krebs.
Protein kinase A antagonizes platelet-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells.
Proc. Natl. Acad. Sci. USA
90:
10300-10304,
1993[Abstract].
11.
Graves, L. M.,
K. E. Bornfeldt,
J. S. Sidhu,
G. M. Argast,
E. W. Raines,
R. Ross,
C. C. Leslie,
and
E. G. Krebs.
Platelet-derived growth factor stimulates protein kinase A through a mitogen-activated protein kinase-dependent pathway in human arterial smooth muscle cells.
J. Biol. Chem.
271:
505-511,
1996[Abstract/Free Full Text].
12.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985[Abstract].
13.
Inoue, S.,
and
S. Kawanishi.
Hydroxyl radical production and human DNA damage induced by ferric nitrilotriacetate and hydrogen peroxide.
Cancer Res.
47:
6522-6527,
1987[Abstract].
14.
Kizaki, Z.,
and
R. G. Thurman.
Stimulation of oxygen uptake by glucagon is oxygen dependent in perfused rat liver.
Am. J. Physiol.
256 (Gastrointest. Liver Physiol. 19):
G369-G376,
1989[Abstract/Free Full Text].
15.
Konger, R. L.,
R. Malaviya,
and
A. P. Pentland.
Growth regulation of primary human ketatinocytes by prostaglandin E receptor EP2 and EP3 subtypes.
Biochim. Biophys. Acta
1401:
221-234,
1998[Medline].
16.
Lehninger, A. L.,
A. Vercesi,
and
E. A. Bababunmi.
Regulation of Ca2+ release from mitochondria by the oxidation reduction state of pyridine nucleotides.
Proc. Natl. Acad. Sci. USA
75:
1690-1694,
1978[Abstract].
17.
Masaracchia, R. A.,
B. E. Kemp,
and
D. A. Walsh.
Histone 4 phosphotransferase activities in proliferating lymphocytes. Partial purification and characterization of an enzyme specific for Ser-47.
J. Biol. Chem.
252:
7109-7117,
1977[Medline].
18.
Pertoft, H.,
and
B. Smedsrod.
Separation and characterization of liver cells.
In: Cell Separation: Methods and Selected Applications, edited by T. G. Pretlow II,
and T. P. Pretlow. New York: Academic, 1987, vol. 4, p. 1-24.
19.
Puschel, G. P.,
C. Kirchner,
A. Schroder,
and
K. Jungermann.
Glycogenolytic and antiglycogenolytic prostaglandin E2 actions in rat hepatocytes are mediated via different signalling pathways.
Eur. J. Biochem.
218:
1083-1089,
1993[Abstract].
20.
Qu, W.,
E. Savier,
and
R. G. Thurman.
Stimulation of monooxygenation and conjugation following liver transplantation in the rat: involvement of Kupffer cells.
Mol. Pharmacol.
41:
1149-1154,
1992[Abstract].
21.
Qu, W.,
Z. Zhong,
M. Goto,
and
R. G. Thurman.
Kupffer cell prostaglandin E2 stimulates parenchymal cell O2 consumption: alcohol and cell-cell communication.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G574-G580,
1996[Abstract/Free Full Text].
22.
Qu, W.,
Z. Zhong,
and
R. G. Thurman.
Stimulation of O2 uptake due to PGE2 is oxygen-dependent in perfused rat liver.
Am. J. Physiol.
275 (Gastrointest. Liver Physiol. 38):
G542-G549,
1998[Abstract/Free Full Text].
23.
Ratto, G. M.,
R. Payne,
W. G. Owen,
and
R. Y. Tsien.
The concentration of cytosolic free calcium in vertebrae rod outer segments measured with fura-2.
J. Neurosci.
8:
3240-3246,
1988[Abstract].
24.
Regan, J. W.,
T. J. Bailey,
D. J. Pepperl,
K. L. Pierce,
A. M. Bogardus,
J. E. Donello,
C. E. Fairbairn,
K. M. Kedzie,
D. F. Woodward,
and
D. W. Gil.
Cloning of a novel human prostaglandin receptor with characteristics of the pharmacologically defined EP2 subtype.
Mol. Pharmacol.
46:
213-220,
1994[Abstract].
25.
Remmer, H.,
J. Schenkman,
R. W. Estabrook,
H. Sasame,
J. Gillette,
S. Narashimhulu,
D. Y. Cooper,
and
O. Rosenthal.
Drug interaction with hepatic microsomal cytochrome.
Mol. Pharmacol.
2:
187-190,
1966[Abstract].
26.
Salm, A. K.,
and
K. D. McCarthy.
Norepinephrine-evoked calcium transients in cultured cerebral type I astroglia.
Glia.
3:
529-538,
1990[Medline].
27.
Sardanelli, A. M.,
Z. T. Dobrova,
S. C. Scacco,
F. Speranza,
and
S. Papa.
Characterization of proteins phosphorylated by the cAMP-dependent protein kinase of bovine heart mitochondria.
FEBS Lett.
377:
470-474,
1996.
28.
Vegesna, R. V.,
and
J. Diamond.
Elevation of cyclic AMP by prostacyclin is accompanied by relaxation of bovine coronary arteries and contraction of rabbit aortic rings.
Eur. J. Pharmacol.
128:
25-31,
1986[Medline].
29.
Walsh, D. A.,
and
D. B. Glass.
Utilization of the inhibitor protein of adenosine cyclic monophosphate-dependent protein kinase, and peptides derived from it, as tools to study adenosine cyclic monophosphate-mediated cellular processes.
Methods Enzymol.
201:
304-316,
1991[Medline].
Am J Physiol Gastroint Liver Physiol 277(5):G1048-G1054
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society