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Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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
![[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB> [(R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R)](F<SUB>o</SUB>/F<SUB>s</SUB>)](/content/vol277/issue5/fulltext/G1048/img001.gif)
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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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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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
cells1, 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).
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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 · h1 · 106
cells1
(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 · h1 · 106
cells1
(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 · min1 · ml1;
PKA + PKI = 8.4 pmol · min1 · ml1;
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.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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, IBMX-treated cells;
, cells without IBMX. IBMX alone had no effect on either cAMP
formation or oxygen consumption. Values are means ± SE
(n = 5).
