1 Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461; 2 Department of Urology, Yamanashi Medical University, Yamanashi 409-3898; and 3 Department of BioMedical Engineering, Tokai University, Kanagawa 259-11, Japan
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ABSTRACT |
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We previously characterized the prostaglandin (PG) transporter PGT as an exchanger in which [3H]PGE2 influx is coupled to the efflux of a countersubstrate. Here, we cultured HeLa cells that stably expressed human PGT under conditions known to favor glycolysis (glucose as a carbon source) or oxidative phosphorylation (glutamine as a carbon source) and studied the effect on PGT-mediated [3H]PGE2 influx. PGT-expressing cells grown in glutamine exhibited a 2- to 4-fold increase in [3H]PGE2 influx compared with the antisense control, whereas cells grown in glucose exhibited a 14-fold increase. In the presence of 10 vs. 25 mM glucose during the uptake, there was a dose-dependent increment in [3H]PGE2 influx. Cis inhibition of [3H]PGE2 influx occurred with lactate at physiological concentrations (apparent Km = 48 ± 12 mM). Preloading with lactate caused a dose-dependent trans stimulation of PGT-mediated [3H]PGE2 uptake, and external lactate caused trans stimulation of PGT-mediated [3H]PGE2 release. Together, these data are consistent with PGT-mediated PG-lactate exchange. Cells engaged in glycolysis would then be poised energetically for prostanoid uptake by means of PGT.
biological transport; organic anion transport; glycolysis
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INTRODUCTION |
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PROSTAGLANDINS (PGS) AND THROMBOXANES have widespread physiological and pathophysiological effects on nearly all cellular processes, including, but not limited to, cardiovascular, gastrointestinal, respiratory, reproductive, renal, and immune systems (56). Because of their broad biological effects, PGs modulate their activity in an autocrine fashion, i.e., they are synthesized by intracellular enzymes at or near their sites of action, exit the cell by simple diffusion (13), and are presented to adjacent PG receptors. Thereafter, extracellular PGs must be metabolized in situ within seconds before they are able to reach the general circulation (17, 21). This loss of biological activity is accomplished through cellular uptake followed by intracellular oxidation (1, 17, 43, 49).
Our laboratory previously identified the novel, broadly expressed PG
transporter PGT, whose substrates include
PGE2, PGF2, PGD2, and
thromboxane-B2 (24). Understanding the
molecular mechanism and driving force of PG transport by PGT is
necessary to identify its role in PG homeostasis for the
organism. PG transport by PGT is Na+, Cl
, and
H+ independent and appears to occur by obligate anion
exchange (13, 24).
Interestingly, in previous studies from our laboratory, concentrative PG uptake was reduced by glycolysis inhibitors and varied with cellular ATP production (13). There is known coupling between glycolysis and PG metabolism. Inhibition of renal papillary glycolysis by various maneuvers increases net PG release, whereas increasing the supply of glucose decreases PG release (22, 57). Our studies showed that PG transport by PGT exhibited a time-dependent overshoot that resulted in transient accumulation of [3H]PGE2 (13), which is consistent with exchange in which [3H]PGE2 influx is coupled to the efflux of a countersubstrate that exhibits a falling outwardly directed gradient. Taken together, the data suggested that PGT-mediated influx of PGs may be coupled to the efflux of an intracellular metabolic end product involved in the generation of ATP by glycolysis.
In the present study, we cultured HeLa cells that stably express PGT under conditions known to favor glycolysis or oxidative phosphorylation and studied the effect on PGT-mediated transport. Our data indicate that the direction and magnitude of PG transport by means of PGT vary directly with the transmembrane concentration gradient of lactate, which is consistent with PG-lactate exchange.
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MATERIALS AND METHODS |
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Materials
Glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, glyceraldehyde-3-phosphate, 1,3,-diphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, and lactate were purchased from Sigma and were the purest grade available. [3H]PGE2 was from DuPont-New England Nuclear.Stable Transfection of PGT in HeLa Cells
HeLa cells (ATCC) were grown in Dulbecco's modified Eagle's medium plus 5% fetal bovine serum and 100 U/ml penicillin-streptomycin (GIBCO). Full-length sense and antisense PGT cDNAs were cloned into the vector pMEP4 (Invitrogen). HeLa cells were transfected with Lipofectin (GIBCO) and pMEP4-antisense human PGT or pMEP4-sense human PGT and cultured in selective media containing hygromycin (600 ug/ml; GIBCO) 48 h later. After 14-18 days, resistant colonies were selected and expanded in selective media.Cell Culture
Stable transfectants were maintained in humidified incubators with 5% CO2 at 37°C in Dulbecco's modified Eagle's medium plus 5% fetal bovine serum and 100 U/ml penicillin-streptomycin. In experiments analyzing the effects of glycolysis on PGT-mediated transport, stable transfectants were grown overnight as monolayers on 35-mm dishes in Dulbecco's modified Eagle's medium with 2 mM glutamine, glutamine plus 25 mM glucose, or glutamine plus 25 mM 2-deoxyglucose supplemented with 5% fetal bovine serum.Transport Assays
Influx measurements. The cell monolayers were washed twice with a balanced salt solution [BSS; (in mM) 135 NaCl, 13 H-HEPES, 13 Na-HEPES, 2.5 CaCl2, 1.2 MgCl2, 0.8 MgSO4, and 5 KCl]. Influx measurements were initiated by the addition of [3H]PGE2 to the flux media (BSS). Influx measurements were carried out at room temperature over 10 min and were terminated by aspiration of the incubation media followed by two rapid washings with ice-cold 5% BSA in BSS and two additional washings with ice-cold BSS. Cells were scraped into 1 ml saline, mixed with a liquid scintillation cocktail (National Diagnostics), and analyzed by liquid scintillation counting. Influx values were calculated as femtomoles per milligram protein per nanomolar concentration [3H]PGE2 and expressed as means ± SE from duplicate monolayers. In some experiments, 10-min uptakes were performed in the presence of BSS vs. BSS plus 10 or 25 mM glucose.
Cis-inhibition.
In cis-inhibition studies, uptakes were performed in the
presence of unlabeled substrates (glucose-6-phosphate,
fructose-6-phosphate, fructose-1,6-diphosphate,
glyceraldehyde-3-phosphate, 1,3,-diphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate,
pyruvate, and lactate). Candidate substrates were added at 10 mM
concentrations during [3H]PGE2 uptake. The
half-maximal inhibition constant (K1/2)
was determined by relating the uninhibited uptake rate to the inhibited uptake rate calculated from the relationship
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Trans-stimulation. In some experiments, stable transfectants were incubated for 15 min in varying concentrations of lactate, washed twice, and then subjected to the [3H]PGE2 uptake assay.
In other studies, monolayers were preloaded with [3H]PGE2 for 20 min. Immediately thereafter, cells were washed twice with room temperature BSS to remove adherent [3H]PGE2. At time 0, 1 ml of BSS ± 25 mM lactate was added to each monolayer. At each subsequent time interval (2- to 10-min intervals), the 1 ml of efflux media was removed for scintillation counting and 1 ml of fresh BSS with or without 25 mM lactate was added. At the end of the experiment, the cell monolayers were scraped and analyzed by scintillation counting. The remaining counts per minute in the cells were added to the sum of the efflux counts to estimate the amount of isotope that had been initially loaded. Efflux values were calculated as the percentage ± SE of the total [3H]PGE2 released at 2-, 5-, and 10-min intervals.Metabolites
Lactate and pyruvate concentrations were measured in the extracellular media by using a colorimetric assay on the basis of the enzymatic conversion of lactic acid to pyruvate and hydrogen peroxide by lactate oxidase (Sigma). Pyruvate concentrations were determined by using a colorimetric assay on the basis of the enzymatic conversion of pyruvate and NADH to lactate and NAD by lactate dehydrogenase (Sigma). Intracellular ATP concentrations of monolayers grown in media with or without glucose were determined by using an ATP assay (Sigma) on the basis of the enzymatic reaction described by Bucher (12). ![]() |
RESULTS |
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Our laboratory previously reported that the PG transporter PGT is probably an obligatory, electrogenic anion exchanger (13). Moreover, our laboratory demonstrated that transport is reduced by inhibiting oxidative phosphorylation and glycolysis, suggesting that the countersubstrate(s) may be a product(s) of intermediary metabolism (13). On the basis of these observations, we cultured HeLa cells that stably expressed human PGT under conditions known to favor glycolysis or oxidative phosphorylation, and then we studied the effect on PGT-mediated [3H]PGE2 influx.
HeLa cells stably transfected with pMEP4-antisense human PGT showed virtually no uptake of [3H]PGE2, which is consistent with previous data from our laboratory (13, 33, 45) and others (4, 8) that PGs at physiological pH enter cells poorly using simple diffusion. In contrast, HeLa cells transfected with pMEP4-sense human PGT showed a 27-fold increase in [3H]PGE2 uptake over that in antisense control (data not shown). All subsequent experiments were performed with the stably expressing sense PGT HeLa cells.
It has been well established that cells in culture can utilize either
glutamine or glucose to provide cell energy, depending on the
availability of the respective substrates (9, 30, 46, 62).
To test the hypothesis that PGT-mediated transport is dependent on
metabolism, we examined the degree to which transport is dependent on
oxidative phosphorylation or glycolysis by incubating our stable
transfectants overnight in media containing 2 mM glutamine with or
without 25 mM glucose. Changing the pattern of metabolism had no effect
on cell morphology; however, the cell-doubling times were ~33 h with
glutamine and 24 h with glutamine plus glucose. No significant
difference in the intracellular ATP concentrations were noted (data not
shown), which is consistent with reports in the literature
(46). However, there was a marked difference in the
ability of PGT-expressing PGT cells to accumulate
[3H]PGE2 as a function of glycolysis. As
shown in Fig. 1, PGT-expressing cells
grown in glutamine plus 25 mM glucose showed a 14-fold increase in [3H]PGE2 influx compared with
PGT-expressing cells grown in glutamine without glucose or in glutamine
with 25 mM 2-deoxyglucose. Similarly, [3H]PGE2 influx in the absence or presence of
10 or 25 mM glucose during 10-min transport assays showed a
dose-dependent increment (Fig. 2). In
contrast, 10-min uptakes in the presence of 10 or 25 mM 2-deoxyglucose,
as an osmotic control, were not different from control uptake (Fig. 1).
Taken together, the data indicate that the rate of
[3H]PGE2 influx appears to be influenced by
the rate of glycolysis.
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To test intermediary metabolites as candidate substrates, we performed
cis-inhibition experiments by adding metabolites to the
outside of the cell during [3H]PGE2 influx.
External substrates other than PGE2 would be expected to
reorient the exchanger toward the cytoplasm, rendering it unavailable to bind and internalize external tracer PGE2. As shown in
Table 1, several glycolytic products
inhibited [3H]PGE2 influx; however, only
lactate inhibited [3H]PGE2 influx at a
concentration that might be approached in the cytoplasm. In accordance
with this concept, in cells grown in glutamine overnight, the lactate
concentration of the external media (which is likely in equilibrium
with the cytoplasm at that point) was 3.5 ± 0.5 vs. 29 ± 6.1 mM in cells grown in glutamine plus glucose. This sevenfold
increase in lactate concentration compares favorably with the sixfold
increase in [3H]PGE2 uptake in cells grown in
the presence of glucose. In contrast, medium pyruvate concentrations
were similar in the two groups (119 ± 13 vs. 123 ± 18 µM). We cannot exclude the possibility that under nonglycolytic
conditions, metabolites other than the lactate shown in Table 1 might
serve as substrates for PGT.
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If lactate serves as the countersubstrate for PGE2 on the
PGT anion exchanger, then the rate of lactate efflux should vary with
the rate of PGE2 accumulation. As demonstrated in Fig.
3, net [3H]PGE2
uptake decreased pari passu with the rate of lactate release over time,
suggesting that PGT-mediated transport is dependent on intracellular
lactate. This is consistent with coupling between [3H]PGE2 uptake and lactate efflux by means
of PGT.
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If lactate is transported by PGT, it should trans accelerate
PGE2 transport, i.e., cytoplasmic lactate would be expected
to reorient the exchanger toward the extracellular space, rendering it
more available to bind and internalize external tracer
PGE2. To test this, we incubated the stable transfectants
overnight in glutamine media so that the intracellular lactate
concentrations before the experiment were low. Then, we incubated the
cells in BSS in the absence or presence of 10 or 25 mM lactate for 15 min before the [3H]PGE2 transport assay. As
shown in Fig. 4, this produced a
dose-dependent increase in [3H]PGE2 uptake,
i.e., lactate trans stimulated
[3H]PGE2 uptake.
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The converse experiments were also performed. Stable transfectants were
loaded with [3H]PGE2, and the rate of
[3H]PGE2 efflux was measured in the absence
and presence of extracellular lactate. As shown in Table
2, trans lactate caused a
significant increase in [3H]PGE2 efflux,
consistent with PGT-mediated lactate-PGE2 anion exchange.
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DISCUSSION |
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We previously found that PGT-mediated PG uptake varied directly with intracellular ATP concentration (13). However, as PGT demonstrates no ATP binding motifs and has no homology to the P-type ATPases, we concluded that ATP is probably indirectly involved in PG transport. We therefore hypothesized that production of an intracellular countersubstrate coupled to PG uptake may be dependent on cellular metabolism. In the present study, we established HeLa cells that stably expressed human PGT and studied the effect of altering glucose metabolism on PGT-mediated [3H]PGE2 uptake. We favored oxidative phosphorylation or glycolysis by supplying cells with glutamine or glutamine plus glucose, respectively, in an overnight incubation. We found that the presence or absence of glucose in the culture media does not result in significant changes in ATP levels. These results are in accordance with those of Reitzer et al. (46), who also found no change in intracellular citric acid intermediates with these maneuvers. Nonetheless, this maneuver results in extremely variable levels of glycolytic intermediates (46). Our data clearly indicate that substantial PG influx could not be supported in the absence of glycolysis. On the other hand, acute exposure of cells to glucose for only 10 min during the transport assay caused a dose-dependent increase in PGT-mediated PG uptake, which is consistent with the coupling of transport to glucose metabolism.
Several results presented here support intracellular lactate as the driving force for prostanoid uptake by means of PGT. First, the degree of increase in lactate concentration in cells cultured in glucose was similar to the degree of increase in PG uptake. Second, the time-dependent net accumulation of [3H]PGE2 varied directly with the rate of lactate efflux. Third, preloading cells with lactate resulted in a dose-dependent augmentation of subsequent [3H]PGE2 influx. Taken together, these data provide strong evidence that intracellular lactate drives PGT-mediated PG uptake.
We hypothesize that lactate is coupled to PG uptake by means of lactate-PG exchange. Several glycolytic metabolites cis inhibited PG influx; however, the Km for lactate is closer to known cytoplasmic lactate levels than those for the other metabolites (7, 19, 34, 48, 52, 54). Indeed, the medium lactate concentration of cells cultured in the presence and absence of glucose was 29 ± 8.5 vs. 3.5 ± 0.7 mM, respectively. On the assumption that the cytoplasmic and medium lactate concentrations are at equilibrium after overnight incubation, these data mean that the lactate concentration within cells was well within the range to serve as a substrate for PGT. In contrast, pyruvate was present at low levels in cells cultured with and without glucose [medium (pyruvate) = 119 vs. 123 µM, respectively], which argued against it being a substrate. Further evidence for lactate-PG exchange is the trans acceleration of [3H]PGE2 efflux by the presence of external lactate; i.e., an inwardly directed lactate gradient results in lactate influx in exchange for PG efflux.
Lactate production under aerobic conditions has been described in many tissues, including lung (28, 32, 40, 47), kidney (38, 60), brain (36, 42), vascular smooth muscle (5, 35, 41), heart (2, 20), and skeletal muscle (11, 23). The presence of outwardly directed lactate gradients in diverse cell types suggests that PGT is poised for PG uptake rather than for release in vivo. Accordingly, using a number of in vitro expression systems, we have failed to demonstrate augmented PG release from cells expressing PGT; on the other hand, we observed that expression of PGT at the plasma membrane results in less net PG release compared with controls (Chan BS, Bao Y, and Schuster VL, unpublished observations).
Taken together, these results suggest that PGT may be involved in reuptake of newly synthesized PGs. This model is similar to that of synaptic signaling, in which plasma membrane neurotransmitter transporters regulate extracellular neurotransmitter levels by uptake. Both prostanoids and neurotransmitters are synthesized and released at their sites of action, where they activate cell surface receptors. In the case of neurotransmission, termination of signaling is accomplished by local reuptake by high-affinity transporters (25, 26, 61). Here, we propose that net release of PGs is controlled by PGT-mediated reuptake within or near cells that release PGs. In concordance with this hypothesis, recent data from our laboratory have localized PGT to cells known to synthesize and release PGs (3).
A reuptake model can explain two previously puzzling papers that showed that inhibiting glycolysis in renal papillae increases the rate of PGE2 release. Herman et al. (22) found that incubation of renal papillae with 2-deoxyglucose or the glycolytic inhibitor NaF increased net PGE2 release. Similarly, Tannenbaum et al. (57) found that increasing amounts of glucose in the buffer suppressed net PGE2 release. One explanation for these findings is that glycolysis promotes PG reuptake by PGT, resulting in a reduction in net PG release.
This hypothesis has ramifications for control of circulation and has potential for explaining vasomotor control during tissue ischemia. Occlusion of blood flow causes release of PGs and PG-mediated vasodilatation (6, 10, 15, 58). In our model, an increase in extracellular lactate in ischemic tissue would create an adverse gradient for PGT-mediated PG reuptake into cells such that the net release of PGs would be augmented.
In addition, Kawamura et al. (27) showed that the
antiapoptotic effect of PGE in PC-12 cells requires PG uptake by
means of PGT. This raises the possibility that extracellular PGs are taken up by cells to signal cell proliferation. In the case of some
malignancies, cell proliferation involves abrogation of
apoptosis (14, 39, 44, 51, 59). Tumor cells
generate large amounts of lactic acid under aerobic conditions
(18, 29, 55), thereby creating a favorable gradient for PG
uptake and any associated antiapoptosis. In addition, smooth
muscle cell proliferation, as observed in atherosclerosis, pulmonary
hypertension, and venous pathologies, appears to be mediated, in part,
by PGD2, PGF2, and PGE (31, 50,
53). This mitogenic response is triggered by exposure of
endothelial cells to hypoxia, suggesting that the pro-proliferative
effect of PGs may be under metabolic control by means of PGT-mediated
uptake into the cell.
In summary, our data demonstrate that PGT-mediated uptake of PGs is dependent on glycolysis and that transport is coupled to lactate by a mechanism consistent with lactate-PG exchange. The present model supports a role for PGT in the uptake of PGs in vivo and provides a molecular explanation for events in which metabolic derangement affects PG release and activity.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-49688 and KO8-DK-02492.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. S. Chan, Renal Div., Albert Einstein College of Medicine, Ullmann Bldg., Rm. 615, 1300 Morris Park Ave., Bronx, NY 10461.
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. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00151.2001
Received 15 May 2001; accepted in final form 26 December 2001.
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