Regulation of mitochondrial glutamine/glutamate metabolism by
glutamate transport: studies with 15N
Tomas
Welbourne1 and
Itzhak
Nissim2
1 Department of Molecular and Cellular Physiology, Louisiana
State University Medical Center, Shreveport, Louisiana 71130; and
2 Division of Child Development and Rehabilitation, Department
of Pediatrics, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
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ABSTRACT |
We focused on the role
of plasma membrane glutamate uptake in modulating the intracellular
glutaminase (GA) and glutamate dehydrogenase (GDH) flux and in
determining the fate of the intracellular glutamate in the proximal
tubule-like LLC-PK1-F+ cell line. We used
high-affinity glutamate transport inhibitors D-aspartate
(D-Asp) and DL-threo-
-hydroxyaspartate (THA)
to block extracellular uptake and then used
[15N]glutamate or [2-15N]glutamine to
follow the metabolic fate and distribution of glutamine and glutamate.
In monolayers incubated with [2-15N]glutamine (99 atom
%excess), glutamine and glutamate equilibrated throughout the intra-
and extracellular compartments. In the presence of 5 mM
D-Asp and 0.5 mM THA, glutamine distribution remained unchanged, but the intracellular glutamate enrichment decreased by 33%
(P < 0.05) as the extracellular enrichment increased
by 39% (P < 0.005). With glutamate uptake blocked,
intracellular glutamate concentration decreased by 37%
(P < 0.0001), in contrast to intracellular glutamine
concentration, which remained unchanged. Both glutamine disappearance
from the media and the estimated intracellular GA flux increased with
the fall in the intracellular glutamate concentration. The labeled
glutamate and NH
formed from
[2-15N]glutamine and recovered in the media increased 12- and 3-fold, respectively, consistent with accelerated GA and GDH flux.
However, labeled alanine formation was reduced by 37%, indicating
inhibition of transamination. Although both D-Asp and THA
alone accelerated the GA and GDH flux, only THA inhibited
transamination. These results are consistent with glutamate transport
both regulating and being regulated by glutamine and glutamate
metabolism in epithelial cells.
glutamate uptake; X
; D-aspartate; DL-threo-
-hydroxyaspartate; L-[2-15N]glutamine; L-[15N]glutamate; 15NH
; L-[15N]alanine
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INTRODUCTION |
GLUTAMINE IS AN
ESSENTIAL amino acid for growth of cells in culture
(10) as well as an important oxidative fuel for cells grown in culture (16, 30) and in intact functioning organs (28, 37). The NH
nitrogen supports both
hepatic ureagenesis (24) and renal ammoniagenesis in vivo (32). The rate-limiting step in intracellular glutamine
utilization via the oxidative pathway is the deamidation reaction (Fig.
1A, reaction
4), catalyzed by phosphate-dependent glutaminase (GA) localized to the inner mitochondrial membrane (7, 16, 30, 32). Recent studies in pig kidney mitochondria have demonstrated that the functional GA activity is expressed on the cytosolic surface
of the inner membrane (17) and represents the dimeric form
of the enzyme (27). Consequently, the products formed in this reaction, glutamate and NH
, are released
directly into the cytosolic compartment (Fig. 1A). To further metabolize glutamate, it must undergo transamination
(reaction 6) to alanine (32) and
-ketoglutarate and/or deamination (reaction 7) to
NH
(32) and
-ketoglutarate. The
deamination reaction is localized to the mitochondrial matrix compartment (16, 30), while the transamination reaction
can occur in both the cytosolic and mitochondrial compartments
(19). Transport of cytosolic glutamate into the
mitochondrial matrix is mediated by two inner membrane glutamate
transporters operating as Glu
/OH
and
Glu
/Asp
exchangers (reaction 3)
coupled to glutamate dehydrogenase (GDH; reaction 7) and
NH
formation and alanine aminotransferase (ALT) and
alanine (reaction 6) formation, respectively (16,
30). The cytosolic glutamate may also be transported out of the
cell through plasma membrane system X
(3) or possibly by the neutral amino acid carrier ASC
(18, 33). Thus plasma membrane (reaction 5) and
mitochondrial membrane glutamate transporters (reaction 3)
compete for the cytosolic glutamate generated from glutamine via the
GA, with alanine, NH
, or glutamate appearing in the
media as products of intracellular glutamine/glutamate metabolism (Fig.
1A).

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Fig. 1.
Glutamate (Glu) fluxes in control
LLC-PK1-F+ monolayers (A) and in the
presence of D-Asp and
DL-threo- -hydroxyaspartate (THA, B).
A: extracellular glutamine (Gln) hydrolyzed by
-glutamyltransferase ( -GT) (reaction 1) to Glu and
NH with the Glu equilibrating with the extracellular
Glu pool. System X -mediated Glu uptake into the
cell (reaction 2) and efflux from the cell (reaction
5) contribute to the extracellular Glu pool turnover.
Intracellular glutaminase (GA, reaction 4) is suppressed by
high cytosolic Glu concentration ([Glu]) as the result of uptake.
Cytosolic Glu equilibrates with intramitochondrial Glu via the inner
membrane exchangers (reactions 3a and 3b) and
with the extracellular pool via the plasma membrane exchanger
(reaction 5). Cytosolic as well as intramitochondrial Glu
can undergo transamination to yield Ala (reaction 6).
Cytosolic Glu can also be effluxed into the media (reaction
5). B: effect of inhibiting plasma and mitochondrial
membrane (Mito) Glu uptake with D-Asp and
L-THA, respectively. Extracellular Glu is blocked from
entering the cell, resulting in reduced cytosolic (Cyto) [Glu] and
accelerated GA flux. The intracellular Glu formed from Gln is effluxed
into the media, undergoes transamination in the cytosol, or enters the
mitochondrial deamination pathway but is blocked by L-THA
from entering the mitochondrial transamination pathway.
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Regulation of glutamine flux through the functional GA is modulated by
the intracellular glutamate concentration (13), which competes with glutamine (31) for the enzyme's reactive
site. Because the functional GA reacts with glutamine and glutamate in
the cytosolic compartment, transport of glutamate into as well as out
of this compartment regulates the GA flux and, hence, glutamine utilization. The uptake of glutamate from the extracellular media (Fig.
1A, reaction 2) has been shown to play an important role in
determining the intracellular glutamate concentration ([Glu]; Fig.
1A) (36). Because the translocated glutamate
and GA share the cytosolic compartment, changes in the availability and
uptake of extracellular glutamate play an important role in regulating intracellular glutamine metabolism. The source of extracellular glutamate is that preformed and present in the media (plasma) plus that
generated by
-glutamyltransferase (
-GT) hydrolysis of
extracellular glutamine (reaction 1) (20, 36).
Under physiological conditions, the extracellular [Glu] ranges from
20 to 50 µM (9), which is within the range of
Michaelis-Menten constants for the cloned X
subtypes expressed in mammalian cells (1). However,
10-fold elevations in plasma [Glu] have been reported in several
pathophysiological conditions (9). Note that de novo
generation of glutamate from extracellular glutamine mediated by
-GT
contributes to the extracellular glutamate pool and ensures continuous
glutamate uptake. The contribution of extracellular glutamate to the
cytosolic pool can be disrupted by blocking the extracellular glutamate
uptake (Fig. 1A, reaction 2) using high-affinity inhibitors
of system X
, D-aspartate and
DL-threo-
-hydroxyaspartate (THA), which competitively inhibit L-glutamate transport across the plasma membrane
(1, 12). Accordingly, blocking glutamate uptake into the
cell should decrease cytosolic [Glu] (Fig. 1B) and
accelerate GA with an increased glutamate formation from cytosolic
glutamine. The glutamate formed in the cytosol should be transaminated
to alanine (reaction 6), transported into the
mitochondrial matrix (reaction 3) forming NH
(reaction 7) or alanine (reaction 6), transported from the cell (reaction 5), or
accumulated with suppression of the GA flux.
To assess the fate of the cytosolic glutamate generated from glutamine,
we incubated LLC-PK1-F+ monolayers in DMEM
containing either 1.8 mM
L-[2-15N]glutamine and 0.5 mM
L-glutamate or 1.8 mM L-glutamine and 0.5 mM
L-[15N]glutamate. Under these conditions,
extracellular glutamate uptake was large, with labeled glutamate
derived from glutamine equilibrating throughout the extra- and
intracellular compartments; a high intracellular [Glu] maintained a
low GA flux. However, in the presence of D-Asp and THA,
labeled glutamate formed from glutamine via the accelerated GA flux did
not equilibrate. Rather, intracellular glutamate decreased, despite a
large increase in GA flux, with the glutamate generated transported out
of the cell, where it accumulated, or into the mitochondria coupled to
deamination and NH
formation, while transamination
was inhibited.
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MATERIALS AND METHODS |
LLC-PK1-F+ cells were grown to
confluence on 60-mm plastic dishes in DMEM containing (in mM) 1.8 L-glutamine, 0.034 L-glutamate, 28 bicarbonate,
10 pyruvate, and 5 D-glucose, as well as 10% fetal bovine serum (Hyclone, Ogden, UT), in 5% CO2-95%
atmosphere. The F+ strain resembles more closely the
proximal tubule than the parental LLC-PK1 cell line, in
that the F+ strain exhibits gluconeogenesis and expresses
an increased
-GT and phosphate-dependent GA activities
(14) (Fig. 1A, reactions 1 and 4).
All experiments were performed 5-9 days after seeding, with fresh
media exchanged daily. The seed plates were routinely screened for
Mycoplasma, with contamination monitored by the use of a
Mycoplasma PCR primer kit (Stratagene Cloning Systems, La Jolla, CA). Individual seed plates were split into six to eight plates,
with three to four used as controls and three to four used as test plates.
Experimental design.
To assess the distribution of labeled glutamate throughout the
intracellular fluid, monolayers were incubated in either
[15N]glutamate and unlabeled glutamine or
[2-15N]glutamine and unlabeled glutamate. The enrichment
of labeled glutamate in the intracellular fluid was then compared with
that in the extracellular fluid. Similar enrichments in the two
compartments were taken to indicate equilibration of the labeled
glutamate throughout the intracellular compartment. To block the
extracellular glutamate uptake (Fig. 1A, reaction
2) as well as to limit mitochondrial glutamate uptake
(reaction 3b), D-Asp (competitively inhibits plasma membrane glutamate uptake by X
, reaction 1) plus THA (L-THA putatively inhibits
mitochondrial glutamate uptake, reaction 2) was added to the
media. To confirm that most of the extracellular glutamate uptake was
inhibited, the incorporation of [15N]glutamate into
alanine was monitored (reaction 6). To monitor the fate of
extracellular glutamate and glutamate formed intracellularly from
glutamine hydrolysis, monolayers were incubated in DMEM plus 0.5 mM
L-[15N]glutamate (99 atom %excess; Cambridge
Isotope Laboratories, Andover, MA) and 1.8 mM L-glutamine
or DMEM plus 0.5 mM L-glutamate and 1.8 mM
L-[2-15N]glutamine (99 atom %excess;
Cambridge Isotope Laboratories). To test the effect of blocking the
plasma membrane glutamate uptake, monolayers were incubated in the
above media plus 5 mM D-aspartate and 0.5 mM THA. According
to the model, labeled glutamate formed from
[2-15N]glutamine (reaction 4) should be
transaminated to alanine (reaction 6), accumulate in the
cytosol, be transported into the mitochondrial matrix via
reactions 3a and 3b, forming NH
and alanine, respectively, or be transported out of the cell
(reaction 5) and appear in the extracellular media behind
the D-Asp block. Inhibiting the
-GT flux (reaction
1) by adding 0.7 mM AT-125 (acivicin; kindly provided by Dr.
Michaele Christian, National Cancer Institute) to the DMEM containing
D-Asp and THA can differentiate the glutamate contributed
by the
-GT-GA reaction (reaction 1) (20)
from the glutamate transported out of the cell (reaction 5).
To test whether system X
plays a role in the efflux
of intracellular anionic amino acids, monolayers were loaded with
radiolabeled D-Asp, which is a specific substrate for
system X
(12). Besides being a
specific substrate for system X
(12),
D-Asp also has the advantage that it is not metabolized by
the LLC-PK1 cell line (29). Thus we used
radiolabeled D-Asp in place of glutamate to determine
whether D-Asp in combination with THA would have a effect
on radiolabeled D-Asp retention different from that of
D-Asp alone. D-[3H]Asp (12 Ci/mmol; NEN, Boston, MA) was loaded into monolayers from DMEM (1 µCi/3 ml media) over 20 min. After 20 min, the plates were washed
three times with ice-cold PBS and then DMEM, DMEM + 5 mM
D-Asp, or DMEM + 5 mM D-Asp + 0.5 mM
THA were added. The plates were then returned to the incubator for 45 min. After 45 min the media were harvested, the monolayers were washed
three times with ice-cold PBS, and then 1 ml of ice-cold 5% TCA was added. The monolayers were promptly scraped free, transferred to a
homogenizing tube, and homogenized by use of a Polytron (half-speed for
30 s). The TCA homogenates were centrifuged (10,000 g
for 10 min), the supernatant was retained to monitor 3H and
glutamate content by liquid scintillation spectrometry, and the pellet
was dissolved to measure protein content. Protein content of the
various fractions was determined by the dye-binding assay
(5) after the TCA-precipitated pellet was dissolved in 0.2 N NaOH.
Analysis.
After 16 h of incubation in the prescribed media, media samples
were taken and the monolayers were rapidly washed three times with
ice-cold PBS. The washed monolayers and aliquots of the medium were
treated with ice-cold 40% perchloric acid. The concentration of
glutamine, glutamate, alanine, and aspartate and their 15N
enrichment were determined on the neutralized supernatants as previously described (24, 25). Briefly, the amino acids
underwent precolumn derivatization with o-phthaldehyde and
separation by HPLC and fluorescent detection (20).
Analysis of 15N in the amino acids was done by gas
chromatography-mass spectroscopy, as previously described (24,
25). Formation of 15NH
was
determined after conversion of NH
to glutamate
(25). NH
concentration was measured by
the previously described microdiffusion method (36).
Calculations.
The net uptake or release of glutamate and alanine was determined from
the difference in the media glutamate present at the beginning
(preformed) and at the end of incubation. The concentration difference
multiplied by the media volume (4 ml) gave the net amount taken up or
produced and expressed per milligram of protein. The net
NH
production was obtained by subtracting the
NH
formed by the monolayers over the 16-h time course
from DMEM without glutamine from that formed in the presence of
glutamine. Aspartate was not produced or taken up in net amounts. To
calculate the conversion of [15N]glutamine to glutamate
and alanine, the isotopic enrichment (atom %excess) of 15N
in the particular metabolite was multiplied by the amount present and
expressed as nanomoles per milligram of protein (25). The sum of the glutamine converted to alanine and to glutamate was added to
the net NH
formation to obtain the glutamine
metabolized through the two GAs and the transamination and deamination
pathways (32). To obtain the glutamine metabolized via the
intracellular pathways, the sum of glutamate and corresponding NH
formed by
-GT (Fig. 1A, reaction 1)
was subtracted from the total (i.e., NH
+ [15N]Ala + [15N]Glu
).
This difference was then divided by 2 to obtain the estimated GA flux
(reaction 4). Comparisons were made between pairs of control and the glutamate uptake-blocked monolayers (total 12-18 pairs). Significant differences were obtained using the Student's
t-test or ANOVA for multiple group comparisons and a one- or
two-tailed t-table depending on whether a priori directional
changes were postulated on the basis of the model shown in Fig. 1.
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RESULTS |
In control monolayers incubated for 16 h with
[2-15N]glutamine, the extra- and intracellular glutamine
enrichments were similar (79 ± 1 and 75 ± 1 atom %excess,
respectively), indicating that glutamine equilibrates throughout the
extra- and intracellular compartments. The intracellular fluid
enrichment of [15N]glutamate (Fig.
2), formed from labeled glutamine, was
equal to the extracellular glutamate enrichment (23 ± 1 and
21 ± 1 atom %excess, respectively) and approximately one-third
of that of [2-15N]glutamine. In the presence of
D-Asp + THA, [2-15N]glutamine enrichment
is unchanged and similar within the two compartments (81 ± 1 and
74 ± 2 atom %excess, respectively). However, the equilibration
of metabolically generated labeled glutamate no longer exists (Fig. 2).
Compared with the control monolayers, enrichment of the intracellular
glutamate decreases (P < 0.05) by ~33% (from
21 ± 1 to 14 ± 0.5 atom %excess), while that of the
extracellular fluid increases 1.4-fold (from 23 ± 1 to 32 ± 2 atoms %excess, P < 0.005). These results are
consistent with transporter-mediated equilibration of glutamate
throughout the intra- and extracellular compartments. More curious is
the decline in intracellular glutamate enrichment in the presence of
D-Asp + THA, despite glutamate generated by the GA
(Fig. 1A, reaction 4; Fig. 1B) from
labeled glutamine within the cell. This finding suggests that the
extracellular GA (reaction 1) is responsible for the
enrichment of labeled glutamate in the extracellular compartment, the
labeled glutamate formed within the cells is exported (reaction 5) and accumulates behind the block in uptake, or both
extracellular production and efflux are occurring.

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Fig. 2.
[15N]Glu distribution between media and
cells in control and in the presence of D-Asp + THA.
Values are means ± SE from 9 pairs of plates incubated for
16 h in the designated media containing an initial 99 atom
%excess [2-15N]Gln. *Different from control,
P < 0.0001.
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In control monolayers incubated with media containing
[15N]glutamate (Fig. 3),
the intracellular enrichment was 38% (P < 0.02) higher than the extracellular enrichment (18 ± 1 vs. 13 ± 1 atom %excess). In the presence of D-Asp and THA,
[15N]glutamate was 3.8-fold enriched in the extracellular
compartment, while intracellular enrichment decreased 70%
(P < 0.0005) compared with the control. These results
show that when the source of labeled glutamate is the extracellular
compartment, inhibiting high-affinity glutamate transport prevents
equilibration between the two compartments with the expected enrichment
of the extracellular [15N]glutamate and a marked decrease
in the intracellular [15N]glutamate. Furthermore, the
decrease in the extracellular [15N]glutamate enrichment
from 99 to 49 ± 3 atom %excess represents the addition of 969 nmol of unlabeled glutamate. To confirm that the extracellular
glutamate uptake was effectively blocked by 5 mM D-Asp and
0.5 mM THA, the formation of alanine from the
[15N]glutamate was determined after 16 h of
incubation. Under these conditions, the alanine formation from labeled
glutamate decreased 87 ± 6% (P < 0.0001) from
236 ± 19 nmol/16 h in the control monolayers to 31 ± 11 nmol/16 h in the presence of D-Asp + THA
(n = 6 pairs of plates). Thus most, if not all, of the
extracellular glutamate uptake is blocked, with the decline in
extracellular enrichment representing addition of unlabeled glutamate.

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Fig. 3.
[15N]Glu distribution between media and
cells in control and in the presence of D-Asp + THA.
Values are means ± SE from 6 pairs of plates incubated for
16 h in the designated media containing an initial 99 atom
%excess [15N]Glu. *Different from media,
P < 0.05; **different from control, P < 0.001.
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To determine the contribution of the extracellular GA to the glutamine
hydrolysis occurring with plasma membrane glutamate uptake blocked,
-GT activity was inhibited and the accumulated glutamate was
compared with that occurring with
-GT active (Fig. 4). In the control monolayers, there is a
net uptake of extracellular glutamate that reverses to a large
accumulation in the presence of D-Asp + THA (from
991 ± 22 to 683 ± 76 nmol · mg
1 · 16 h
1,
P < 0.0001). Inhibiting the
-GT with acivicin
reduced (P < 0.004) the glutamate accumulating in the
presence of D-Asp + THA by 48% (from 683 ± 76 to 358 ± 32 nmol · mg
1 · 16 h
1). This indicates that 52% of the glutamate
accumulating in the media with the glutamate uptake blocked is derived
from an intracellular source.

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Fig. 4.
Media Glu uptake (bottom) or accumulation
(top) in control and with D-Asp + THA or
D-Asp + THA + acivicin. Values are means ± SE from 12 control and 6 pairs of D-Asp + THA plates
with or without 0.75 mM acivicin. *Different from control; **different
from D-Asp + THA, P < 0.05.
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To demonstrate that blocking glutamate uptake specifically accelerated
intracellular GA flux, monolayers were incubated with [2-15N]glutamine and the 15N-labeled products
were determined as presented in Table 1.
After 16 h of incubation with D-Asp + THA, total
cellular glutamate was decreased 37% (from 182 ± 10 to 114 ± 8 nmol/mg protein, P < 0.0001, n = 18 pairs), The intracellular glutamine content remained unchanged
(48 ± 2 vs. 48 ± 3 nmol/mg protein). Consequently, the intracellular ratio of glutamine to glutamate increases, favoring accelerated glutamine breakdown. The sum of glutamate and alanine produced from the 15N-labeled amino nitrogen of glutamine
plus the total NH
formed increased 61%
(P < 0.001) in the presence of D-Asp + THA (from 1,357 ± 52 to 2,179 ± 57 nmol/mg protein). The
increase in these products resulting from glutamine hydrolysis was
associated with enhanced (P < 0.001) glutamine
disappearance from the media (1,329 ± 83 vs. 1,526 ± 86 nmol · mg
1 · 16 h
1),
consistent with an accelerated GA flux. Recovery of the labeled [2-15N]glutamine as glutamate increased ~12-fold (from
65 ± 6 to 745 ± 60 nmol/16 h, P < 0.0001).
In addition, the total NH
production also increased
(P < 0.0002) from 995 ± 68 to 1,186 ± 96 nmol · mg
1 · 16 h
1.
However, alanine formation from [2-15N]glutamine
decreased (from 298 ± 40 to 252 ± 27 nmol · mg
1 · 16 h
1,
P < 0.05). Nevertheless, the sum of the GA products
clearly increased, although the relative contribution of the extra- and intracellular pathways requires further analysis. The contribution of
the extracellular
-GT activity determined above (Fig. 4) as 325 nmol/mg was doubled to include the NH
and then
subtracted from the control and D-Asp + THA product
total. The total of NH
, alanine, and glutamate formed
from the intracellular pathways was then divided by 2 to obtain the GA
flux in nanomoles per milligram of protein (707 ± 53
2 = 354 ± 26 and 1,530 ± 57
2 = 764 ± 29 nmol/mg). In the control monolayers (Fig.
5), the estimated intracellular GA flux
(Fig. 1A, reaction 4) was approximately equal to the flux through the extracellular GA (reaction 1): 354 ± 26 and 325 ± 105 nmol glutamine hydrolyzed/mg protein, respectively.
However, after the uptake of the extracellular glutamate was blocked,
the estimated intracellular GA flux increased 2.2-fold (from 354 ± 26 to 764 ± 29 nmol glutamine/mg protein, P < 0.001). Note that total NH
production, rather than
15NH
derived from the
[2-15N]glutamine, was used to estimate the GA flux in
these studies.

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Fig. 5.
Extra- and intracellular Gln hydrolysis in control
monolayers and monolayers treated with D-Asp + THA.
Extracellular hydrolysis was taken as the acivicin-inhibitable Glu
formation [ -GT phosphate-independent glutaminase ( -GT-PIG)] in
the presence of D-Asp (D-A) + THA.
Intracellular Gln hydrolysis by phosphate-dependent GA (PDG) is
Glu + Ala formed from labeled Gln + NH 2 (see MATERIALS AND METHODS for calculation). Values are
means ± SE from 9 pairs of plates. *Different from control,
P < 0.001.
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To determine the metabolic fate of intracellular glutamate and to
differentiate the action of D-Asp from that of THA, we
monitored the formation of 15NH
and
[15N]alanine from 15N-labeled amino nitrogen
of glutamine in the presence of each inhibitor alone and in combination
(Fig. 6). In the presence of D-Asp + THA, NH
formed from
glutamate deamination was increased 3.1-fold (P < 0.01). Furthermore, THA added to D-Asp increased
(P < 0.03) the deamination rate compared with
D-Asp alone (575 ± 40 vs. 348 ± 41 nmol/mg).
The alanine formation from 15N-labeled amino nitrogen of
glutamine was reduced (P < 0.03) by THA, indicating
that THA impedes the ALT flux; this effect of THA was also in evidence
in combination with D-Asp (357 ± 36 vs. 590 ± 67 nmol/mg for D-Asp alone, P < 0.05).
Both D-Asp and THA increased the accumulation of
[15N]glutamate in the media, although THA reduced
(P = 0.05) the D-Asp-induced accumulation
(400 ± 60 vs. 575 ± 27 nmol/mg). These results show that
both THA and D-Asp alone increase the GDH flux but that THA
decreases the ALT flux whether alone or in combination with
D-Asp; additionally, THA apparently acts to slow the
D-Asp-accelerated glutamate efflux.

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Fig. 6.
Effect of THA or D-Asp alone and in
combination on [15N]Glu,
15NH and [15N]Ala formation
from the [2-15N]Gln. Monolayers were incubated for
16 h in DMEM, DMEM + 0.5 mM THA, DMEM + 5 mM
D-Asp, or DMEM + 0.5 mM THA + 5 mM
D-Asp (DMEM contains 0.05 mM L-Glu). Values
[isotopic enrichment (atom %excess) × concentration (nmol/mg
protein)] are means ± SE. *Different from controls,
P < 0.05; **different from D-Asp,
P < 0.05.
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The acute effect of D-Asp and THA on efflux pathway(s) was
studied in monolayers preloaded with the nonmetabolizable
D-[3H]Asp and compared with glutamate efflux.
After the monolayers were preloaded with
D-[3H]Asp, they were placed in control DMEM,
DMEM containing 5 mM D-Asp, or DMEM containing 5 mM
D-Asp + 0.5 mM THA for 45 min; then the
D-[3H]Asp retained in the monolayer was
determined (Fig. 7). Compared with the
control monolayers, those exposed to 5 mM D-Asp or 5 mM
D-Asp + 0.5 mM THA retained 31 and 36% less
D-[3H]Asp than the controls (82 ± 16 × 103 vs. 57 ± 20 × 103
and 52 ± 19 × 103 cpm/mg protein after 45 min,
n = 4 pairs, P < 0.01). Compared with
monolayers taken at time 0, the control monolayers taken at
45 min had already lost 43 ± 10% of their
D-[3H]Asp. These findings indicate that
D-Asp and D-Asp + THA accelerate the
efflux of intracellular substrates for system X
and
that a significant efflux occurs even in the normal mix of media amino
acids. In this acute 45-min study, glutamate accumulated in the media
with D-Asp and D-Asp + THA (Fig.
8), in contrast to the controls, which
did not show an accumulation (4.4 ± 0.2 and 2.8 ± 0.4 nmol · mg
1 · min
1 for
D-Asp and D-Asp + THA, respectively, vs.
0.2 ± 0.3 nmol · mg
1 · min
1 for
control, P < 0.0001). The presence of THA reduced
(P < 0.05) the media glutamate accumulation compared
with the presence of D-Asp alone (2.8 ± 0.4 vs.
4.4 ± 0.2 nmol · mg
1 · min
1) and also
decreased (P < 0.01) the formation of alanine compared with D-Asp (11.7 ± 0.7 vs. 16.2 ± 0.3 nmol · mg
1 · min
1,
P < 0.01). As expected from the longer term study
above, THA + D-Asp increased (P < 0.05) the NH
formation over that produced with
D-Asp alone. D-Asp alone and D-Asp + THA reduced the monolayer glutamate content
(134 ± 10 and 131 ± 12 vs. 198 ± 3 nmol/mg protein,
both P < 0.02 vs. control), indicating that THA did
not back up the glutamate in the cytosol. These results suggest that
THA does not reduce glutamate efflux by system X
but acts on a second stereoselective system that also contributes to
extracellular glutamate accumulation under these conditions.

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Fig. 7.
Effect of D-Asp vs. D-Asp + THA on retention of D-[3H]Asp. Monolayers
were loaded with D-[3H]Asp for 20 min, washed
3 times with PBS, and harvested (time 0) or incubated for 45 min in DMEM, DMEM + 5 mM D-Asp, or DMEM + 5 mM
D-Asp + 0.5 mM THA. Values are means ±SE from 4 plates/group. *Different from 45 min, P < 0.05.
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Fig. 8.
Effect of D-Asp vs. D-Asp + THA on Glu, NH , and Ala appearance in the media after
45 min of incubation. Values are means ± SE from 4 plates/group.
*Different from control, P < 0.05; **different from
D-Asp, P < 0.05.
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DISCUSSION |
The objectives of this study were to determine the role of
glutamate transporter activity in intracellular glutamine hydrolysis and in the fate of the intracellular glutamate generated (Fig. 1A). We chose inhibitors of the high-affinity plasma
membrane glutamate transporters to block uptake of extracellular
glutamate and to lower the intracellular glutamate concentration and,
thereby, activate the intracellular GA (Fig. 1B). The
inhibitors chosen did block extracellular glutamate uptake and produced
a prompt and sustained reduction in the intracellular glutamate
content. As a consequence of reducing this potent functional GA
inhibitor (17), hydrolysis of glutamine increased more
than twofold (Fig. 5). These results are consistent with the system
X
-transported glutamate setting the cytosolic
glutamate concentration, which, in turn, acts as a signal
(35) in regulating flux through the functional GA.
Accordingly, plasma membrane glutamate transport regulation of the
functional GA is consistent with localization at the cytosolic
(17), rather than the matrix, surface (7) of
the inner mitochondrial membrane. The second objective was to determine
the fate of the intracellular glutamate formed from glutamine. If the
functional GA was located within the mitochondrial matrix, the
glutamate produced could be deaminated or transaminated or could
accumulate and inhibit the GA flux (16), since
mitochondrial glutamate efflux is limited (30). We show
that the glutamate produced does not accumulate; rather, the
intracellular concentration remains depressed, with a large efflux of
labeled glutamate formed from glutamine from these cells. The failure
of the labeled glutamate to equilibrate throughout the intracellular
compartment indicates that the functional GA activity is in close
proximity to the plasma membrane transporters. A preferential efflux of
intracellular glutamate formed from labeled glutamine, as opposed to
equilibration within the intracellular fluid, has previously been
observed in the brain slices (4). Our findings reveal a
major efflux pathway for glutamate produced from glutamine by the
functional GA.
Chronic metabolic acidosis reduces intracellular glutamate content,
despite a two- to threefold increase in the functional GA flux
(32). The reduced intracellular glutamate is attributed to
enhanced flux through the GDH pathway, in turn promoted by the cellular
acidosis (32). Indeed, in our study the flux through the
intramitochondrial GDH pathway was increased threefold compared with
the control (Fig. 6). Given the matrix localization for GDH (16,
30) and the cytosolic functional GA, glutamate transport into
the mitochondria must be increased to support the observed flux. The
pH-sensitive mitochondrial Glu
/OH
exchanger
couples cytosolic glutamate to the GDH pathway, and since a decrease in
cytosolic (media) pH increases the matrix glutamate concentration
(30) and accelerates the GDH flux (25), it
seems reasonable to ascribe the enhanced deamination to a decrease in
the cytosolic pH. The system X
-mediated glutamate
uptake also delivers an acid load, producing an intracellular acidosis
(15). In the presence of 5 mM D-Asp + 0.5 mM THA, it seems likely that the intracellular pH is depressed
(20), driving the cytosolic glutamate into the GDH
pathway. Accordingly, system X
-mediated glutamate
transport may regulate the rate of intracellular glutamate formation as
well as the fate of the formed glutamate through two different
modalities of the carrier's activity (35).
Curiously, the conversion of glutamate's amino nitrogen to alanine
actually decreases in the presence of THA and D-Asp + THA but not in the presence of D-Asp alone (Table 1, Figs.
6 and 8). Although THA has traditionally been deployed as a
high-affinity glutamate transporter inhibitor, the presence of the
L-isomer may provide for interaction with other
transporters or enzymes, as recently suggested (8). Our
results clearly show that THA inhibits the transamination reaction
involving the amino nitrogen of glutamate by blocking glutamate
transport into the mitochondria (2) or directly inhibiting
the reaction of glutamate with the enzyme (11). There are
two forms of ALT as depicted in Fig. 1: one is present in the cytosol,
and the other is localized to the mitochondrial matrix
(19). Because L-THA is a competitive inhibitor
of mitochondrial glutamate uptake via the
Glu
/Asp
exchanger (2), it
could limit the available glutamate for intramitochondrial alanine
formation. The presence of the cytosolic ALT activity might explain the
reduced alanine formation in the presence of THA. Alternatively, the
reduced alanine formation may reflect L-THA displacing
glutamate from the ALT-reactive site (11). The effect of
THA to reduce alanine formation was previously noted (34),
while the overall energy generated by the mitochondria is maintained by
the increased flux through the deamination pathway (Fig. 6). Further
studies comparing the effects of D- and L-THA are required to confirm the active isomer and the mechanism of action
in inhibiting transamination.
Glutamate metabolism is highly compartmentalized and, therefore,
dependent on translocation between intra- and extracellular compartments (6, 16, 30). In the control monolayers,
labeled glutamate, produced in both compartments, equilibrated
throughout the extra- and intracellular compartments (Fig. 2),
indicating that glutamate transporter activities in the plasma
(6) and mitochondrial (16, 30) membranes are
able to keep pace with the localized glutamate formation. This reveals
a high bidirectional flux of glutamate across the plasma membrane under
these conditions (Fig. 1). Furthermore, essentially all the glutamate
uptake and a significant part of the efflux is dependent on the plasma
membrane system X
activity, since D-Asp
and THA prevented equilibration of labeled glutamate added to the media
(Fig. 3). System X
has been shown to function in
both modes (22), and countertransport activity plays
important homeostatic roles (6). Our results show that the
media labeled glutamate decreased 50%, indicating an influx of
nonlabeled glutamate, since uptake was blocked. The sources of this
nonlabeled glutamate were subsequently shown to be glutamine hydrolyzed
by the extracellular
-GT and glutamate transported out of the cell
(Fig. 4). The intracellular source was further shown through the
15N label to be glutamine hydrolyzed by the functional GA.
Inhibiting system X
-mediated plasma membrane glutamate uptake in the brain led to >30% of labeled extracellular glutamine recovered as labeled extracellular glutamate
(21). These findings suggest that the model presented in
Fig. 1 may have a wider application than epithelial cells.
The surprising large fluxes of glutamate into and out of these cells
indicate a significant cycling of glutamate across the plasma membrane,
with system X
acting as a homoexchanger (Fig. 1).
The large influx was supported by the preformed and the
-GT-generated glutamate as well as by a significant efflux of
intracellular glutamate. Evidence for the efflux pathway in the control
monolayers was the equilibration of labeled glutamate formed from
labeled glutamine throughout the intra- and extracellular compartments;
evidence that system X
was involved in the plasma
membrane cycling of glutamate was the efflux of labeled
D-Asp from preloaded monolayers (Fig. 7) and the disruption
of the influx and acceleration of the efflux by D-Asp + THA (system X
acting as a heteroexchanger)
(22). The apparent function of this putative membrane
cycling is to maintain a high X
activity. The
carrier turnover and, more specifically, the extravagant movement of
ions (3 Na+/1 K+) may act as a regulator of
glycolysis as they do in astrocytes (8). Accordingly, in
epithelia, the formation of ADP, as a by-product of restoring the ionic
gradients, may act to stimulate glycolysis and ATP formation, in turn
coupled to cellular processes, e.g., paracellular permeability
(34, 35). An excess of cycled glutamate is drawn off into
the transamination or deamination pathways depending on the acid/base
conditions. A deficit, on the other hand, accelerates glutamine
hydrolysis supplying the cycled glutamate. Although metabolic acidosis
decreases the extracellular glutamine hydrolysis (35), a
fall in intracellular pH enhances the efflux of glutamate
(26), ensuring a continuous supply for sustained glutamate
uptake. From this perspective, glutamine/glutamate metabolism is
regulated by glutamate transport and, in turn, plays a role in
maintaining an adequate supply of glutamate dedicated to plasma
membrane cycling and its function in cellular processes.
 |
ACKNOWLEDGEMENTS |
We thank Ilana Nissim and Adam Lazarow for excellent technical support.
 |
FOOTNOTES |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-53761 (to I. Nissim) and
Genentech and the Southern Arizona Foundation (to T. Welbourne).
Address for reprint requests and other correspondence: T. Welbourne, Dept. of Molecular and Cellular Physiology, LSUHSC,
Shreveport, LA 71130 (E-mail twelbo{at}lsumc.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 1 September 2000; accepted in final form 21 November 2000.
 |
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