Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32610
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ABSTRACT |
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We utilized HRP.1 cells derived from midgestation rat
placental labyrinth to determine that the primary pathway for glutamate uptake is via system X-(methylamino)isobutyric acid. Immunoblot analysis of the three transport proteins previously associated with system
X
placenta; aspartate
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INTRODUCTION |
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THE PLACENTA PLAYS an integral role not only in the supply of nutrients to the fetus but also in the maintenance of pregnancy (13). Accordingly, the absorption of glutamate by the placenta is thought to be integral to fetal well being and development, at least in part because of the maternal/fetal "glutamine-glutamate cycle." In this cycle, which has been carefully defined in the ovine model, maternal glutamine is transferred across the placenta to the fetal circulation, extracted by the fetal liver, and then deaminated for nitrogen utilization and release of glutamate (2, 22, 26, 31, 45). Glutamate derived from the fetus is then returned to the placenta, where the majority is oxidized to CO2 and H2O.
We previously characterized the systems responsible for the transport
of glutamate within the rodent placental labyrinth. System
X
Although invaluable for studies such as those denoted above, in vivo
models are of limited usefulness for the study of transporter regulation, especially because it may be linked to cellular metabolic status. Upregulation of system X-ketoglutarate through the action of glutamate dehydrogenase or an
aminotransferase. These activities have been demonstrated in the
placenta (3, 39).
The availability of placental cell type-specific cell lines greatly facilitates studies of the relationships among glutamate transport, transport regulation, and transporter expression (6). The rat placenta is composed of different anatomic and functional regions, foremost of which is the junctional zone, composed largely of spongiotrophoblast cells, important in placental hormone production, and the labyrinth zone, thought to be the area in which the bulk of maternal fetal nutrient transfer occurs. Several rat placental cell lines have been derived from midgestation rat placenta and are extensively characterized by Hunt et al. (16, 17). The HRP.1 line, derived from explants of days 11-12 placental labyrinth, exhibits selective characteristics of labyrinth cells. Specifically, it expresses transferrin receptor and alkaline phosphatase, characteristics of labyrinth cells. HRP.1 cells also secrete placental lactogen 2 (consistent with labryinthine giant cells) but do not form syncytia (16, 17, 41). Because of its rodent origins, derivation from, and similarity to labyrinth cells, we have chosen the HRP.1 cell line for our studies of anionic amino acid transport regulation within a placental cell line.
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METHODS |
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Chemicals. [3H]glutamic acid was obtained from American Radiolabeled Chemicals (St. Louis, MO). All other chemicals were of reagent grade or of the highest grade commercially available. The EAAC1 antibody was made as previously described (28). GLT1 and GLAST1 antibodies were the kind gifts of Jeffrey Rothstein (Johns Hopkins University). The modified 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was obtained from Sigma (St. Louis, MO). 4-[3-(4-Iodophenyl)-2(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate (WST-1) assay kits were obtained from Boehringer Mannheim.
Cell culture. HRP.1 cells were the kind gift of M. J. Soares. They were grown as described by Hunt et al. (16, 17) except that NCTC-135 media and 10% fetal bovine serum were used. Amino acid depletion experiments were performed using Selectamine media with the addition of the designated amino acid in a concentration of 1 mM. Dialyzed serum was used for all studies. Studies described were performed with cells at ~80% confluence unless otherwise noted. Transport was performed as described below after plating cells into 24-well trays.
Enzyme assays. Transaminase activity was assayed with a kit obtained from Sigma. Glutamate dehydrogenase and glutaminase assays were performed according to previously published protocols (7, 29, 37).
Transport.
Cells were seeded into 24-well dishes as described earlier, and whole
cell transport was performed as previously described (20).
Transport was initiated by replacing depletion (amino acid free) buffer
(2 × 15 min) with Na+-containing Krebs-Ringer
phosphate or Na+-free (choline KRP) buffers that contained
the appropriate amount of radiolabeled (10 µCi/ml) amino acids, and,
when indicated, inhibitors. After the appropriate time interval, which
was typically <60 s, uptake was terminated by four rinses of 4°C
choline KRP (2 ml/well). After air drying, cellular protein was
precipitated with 10% TCA, and the supernatant radioactivity was
analyzed by liquid scintillation counting. Proteins were solubilized in
0.2 N NaOH/0.2% SDS and analyzed for total cellular protein. Uptake velocities (uptake · mg
protein1 · min
1) were reported as
means ± SE unless otherwise noted. Derived (i.e.,
sodium-dependent, starvation-induced) velocities were obtained by
subtracting uptakes in the absence of Na+/presence of
inhibitor from that in the presence of Na+ or absence of
inhibitor. SE were then derived in the usual fashion.
Biotinylation. The method utilized for biotinylation of plasma membrane proteins was that of Sims and colleagues (42). Cells were rinsed with PBS containing 0.1 mM Ca2+ and 1.0 mM Mg2+ and were then incubated with biotin solution (sulfo-N-hydroxysuccinimide-biotin, 1 mg/ml in PBS-Ca2+/Mg2+) for 20 min. Biotin solution was aspirated, and the cells were washed two times and then incubated for 30 min with PBS-Ca2+/Mg2+ containing 100 mM glycine. After two additional washes, the cells were incubated with RIPA/lysis buffer (100 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% SDS, and 1% sodium deoxycholate) for 30 min at 4°C. Lysates were subsequently centrifuged at 20,000 g, and supernatants were incubated with avidin-conjugated beads for 1 h at room temperature. After centrifugation at 20,000 g for 15 min, the pellet was resuspended in RIPA/lysis buffer, washed four times, and then brought up in Laemmli buffer (62.5 mM Tris · HCl, pH 6.8, 2% SDS, 20% glycerol, and 5% 2-mercaptoethanol). After a 30-min incubation, the mixture was centrifuged at 20,000 g for 5 min, and the supernatant (biotinylated fraction) was used for immunoblotting.
Western analysis. Protein aliquots (50 µg/lane) were electrophoresed on 7.5% SDS-PAGE by the method of Laemmli (21). Proteins were electrotransferred to a 0.45-µm nitrocellulose membrane, and blots were probed with the specified amount/dilution of antibody. Immunoreactive bands were detected with protein A conjugated to horseradish peroxidase (HRP) or HRP-conjugated secondary antibody, as appropriate. Visualization was performed with an enhanced chemiluminescence kit. These antibodies were previously validated and found to be specific through the use of preincubation of primary antibody with the appropriate peptide or fusion protein against which each antibody was made, with subsequent disappearance of visible bands. Densitometry was performed using NIH software.
Data analysis. Kinetic analyses were performed by using computer-assisted least-squares fits of data points with the EnzFitter computer program (Elsevier-Biosoft). Data were examined for the presence of multiple transport systems both graphically and with the aid of a computer. Differences between groups were determined with Student's two-tailed t-test or by the test of differences of means (in the case of derived values, such as Na+-dependent uptakes). Data were expressed as means ± SE unless otherwise specified.
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RESULTS |
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Glutamate uptake into apical and basal membrane vesicles derived
from rat placental labyrinth was previously demonstrated to occur via
system X
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Uptake via system X
We next examined the kinetics of both Na+-dependent and
system X
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System X-(methylamino)isobutyric acid (MeAIB), a nonmetabolizable amino acid
analog substrate of system A, had no effect at any tested time point.
Although confluence was visually unaffected by the treatments described
above, protein contents were significantly decreased in each treatment
group compared with amino acid-replete controls (data not shown),
raising the possibility that the results observed were the consequence of diminished confluence rather than substrate deprivation. Conversely, protein contents in the glutamate-, glutamine-, and MeAIB-replete groups were virtually identical in each set (6, 24, and 48 h). Given the large range in uptake shown in these groups, from glutamate replete, which was equivalent to the amino acid-replete control, to
MeAIB, which was equivalent to the amino acid-depleted group, it seems
unlikely that the observed results were solely attributable to
differences in confluence.
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Five amino acid transporters capable of glutamate transport consistent
with system X
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To begin to define the role of individual transport proteins in the
transfer of glutamate into HRP.1 cells, we examined glutamate uptake in
the presence of varied inhibitors. System X-hydroxyaspartate and
L-trans-2,4-pyrolidine dicarboxylic acid but not
by dihydrokainate (Fig. 5). This
inhibition profile suggests that despite its presence within the cell,
GLT1 was not an important component of system X
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We next examined the role of intracellular anionic amino acid
metabolism on the regulation of anionic amino acid uptake into HRP.1
cells. Glutaminase, aminotransferase, and glutamate dehydrogenase activities were assessed in HRP.1 cells utilizing previously validated assays [glutamate dehydrogenase (29), aspartate
aminotransferase (Sigma kit), and glutaminase (7, 37)].
Both glutaminase and aminotransferase activities were present, and
conditions for inhibition of activity with
6-diazo-5-oxo-L-norleucine (DON) and aminooxyacetic acid
(AOA) were established (data not shown). DON is an established inhibitor of phosphate-dependent glutaminase activity, whereas AOA is
an established inhibitor of aminotransferases in both human and rat
placenta (4, 9, 34, 37). Glutamate dehydrogenase activity
could not be demonstrated in HRP.1 cells. Therefore, the impact of DON
and AOA on glutamate uptake into HRP.1 cells was examined in the
presence/absence of specific amino acids. Neither AOA (10 mM) nor DON
(2.5 mM), by themselves, had a significant impact on uptake under any
tested condition (data not shown). As shown in Fig.
8, however, the inhibitors in combination
had a significant impact on anionic amino acid uptake. In the presence of amino acids, the combination of inhibitors diminished glutamate uptake. This change was not associated with either a decrease in
protein or in WST-1 assay (measure of metabolically active cells)
results, suggesting that the combination of inhibitors was not
associated with a decrease in cell viability. Uptake was also decreased
when glutamate, aspartate, or glutamine (not shown) was selectively
deleted from the media. In amino acid-free media, inhibitors were
associated with a trend toward enhanced glutamate uptake; however,
these differences did not reach statistical significance. The presence
of inhibitors in addition to either glutamate or glutamine (not shown)
elicited a significant increase in uptake. Conversely, in the presence
of aspartate, glutamate uptake was diminished in the presence of
inhibitors. Asparagine, in the absence of other amino acids, was
insufficient to suppress glutamate uptake to the level noted in the
presence of amino acids, glutamate alone, or aspartate alone. In the
presence of inhibitors, however, like aspartate and unlike glutamate or
glutamine, uptake was diminished compared with that in the absence of
inhibitors. Uptake of glutamate into cells whose media lacked only
asparagine was also diminished in the presence of inhibitors.
Interestingly, this effect was lost if both aspartate and asparagine
were depleted. Uptake in this case (presence of inhibitors) was not
significantly different from that noted in the absence of amino acids.
Together, these data suggest that uptake activity was regulated
primarily by the presence of aspartate, rather than glutamate, and that
under normal conditions, sufficient intracellular aspartate can be
derived from either glutamate or asparagine.
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DISCUSSION |
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Glutamate is produced by the fetal liver from glutamine and is
subsequently taken up by the placenta, wherein it is thought to be an
important substrate for placental energy production (31, 45). We have previously demonstrated the presence of system X
As an initial step in this process, we demonstrated that system
X
We have previously shown that at least three transporters capable of anionic amino acid transport are present within the rat placental labyrinth. We explored the expression of these transport proteins in response to amino acid depletion. In agreement with prior findings by McGivan and colleagues (30, 35, 38), there was no increase in EAAC1 expression at either 6 or 24 h in amino acid-starved cells. Indeed, expression was actually suppressed by the addition of glutamate and glutamine to the media at 6 h. Expression was increased at 48 h, although this increase reached significance only when glutamate was in the media. GLAST1 expression was qualitatively similar to that of EAAC1. Although GLT1 expression was increased at 6 h, expression decreased with increasing duration of amino acid starvation. These data seemed initially to conflict with our demonstration that a significant portion of anionic amino acid uptake induced by starvation is inhibitable by dihydrokainate, and, therefore, may be attributable to GLT1 expression. The subsequent biotinylation data, conversely, demonstrated that although total GLT1 expression may have fallen with amino acid depletion, the cell surface expression of this protein rises under the same conditions by two- to threefold.
Finally, we explored the impact of intracellular anionic amino acid metabolism on anionic amino acid uptake. Inhibition of glutaminase and aminotransferase activities in the presence of amino acids inhibited glutamate uptake into HRP.1 cells. Uptake continued to be downregulated despite the absence of glutamate, aspartate, or glutamate and aspartate (data not shown), suggesting that either these amino acids do not, of themselves, regulate transporter activity or that intracellular synthesis of these amino acids was not completely inhibited. The increase in uptake associated with the addition of inhibitors in the amino acid-depleted/glutamate-sufficient condition suggests that the products of glutamate metabolism, rather than glutamate per se, were required for the normalization of uptake demonstrated in the absence of inhibitors compared with the amino acid-replete condition. Conversely, the decrease in uptake demonstrated in the amino acid-depleted/aspartate-replete condition in the presence of inhibitors suggests that aspartate, or a metabolite thereof, might participate directly in uptake regulation. Aspartate may be synthesized/degraded via the action of transaminases. It may also participate in the urea cycle, via conversion to arginosuccinate. Finally, asparagine and aspartate may be interconverted by asparaginase or asparagine synthetase activities (44). Our finding that asparagine, like aspartate, sufficiency was associated with inhibition of glutamate uptake in the presence of metabolic inhibitors suggests that this pathway might be of importance in HRP.1 cells. We have, therefore, demonstrated, for what is to our knowledge the first time, that anionic transport activity, at least in HRP.1 cells derived from the rodent placenta, appears to be regulated preferentially by aspartate rather than glutamate. This is perhaps not surprising given the key role of aspartate in cellular metabolic pathways, including the urea cycle, citric acid cycle, and nucleic acid synthesis. We cannot rule out the possibility that other amino acids not tested in this study might have a similar effect.
In summary, HRP.1 cells derived from rat placenta demonstrate system
X
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Child Health and Human Development Grant HD-29934.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. Novak, Dept. of Pediatrics, Division of Pediatric Gastroenterology and Nutrition, Univ. of Florida College of Medicine, PO Box 100296, Gainesville, FL 32610 (E-mail: novakda{at}peds.ufl.edu).
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.
Received 20 February 2001; accepted in final form 30 April 2001.
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