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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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>)
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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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.



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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.



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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).


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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).



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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).


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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.


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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.


                              
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Table 1.   Effect of protein kinase A on mitochondrial respiration


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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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.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Am J Physiol Gastroint Liver Physiol 277(5):G1048-G1054
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