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INTRODUCTION |
Virtually every field of physiology has been influenced by
nitrogen monoxide (NO),1 a
small, relatively unstable, potentially toxic, diatomic free radical
gas, that is produced in a diverse variety of mammalian cells (for
reviews see Refs. 1-3). NO has a physiological role as a short lived
messenger molecule and has two principal functions in cells,
servoregulation and cytotoxicity. Considering servoregulation, NO is
produced in small amounts under physiological conditions and mediates
vasorelaxation, regulates blood pressure, controls the adhesion and
aggregation of platelets and neutrophils, and is involved in
neurotransmission. Most of these actions are mediated through the
binding of NO to iron in the heme prosthetic group of soluble guanylate
cyclase (1, 3). Indeed, the high affinity of NO for iron and other
metal ions is a well known branch of coordination chemistry (2).
The importance of iron in mediating the functions of NO is also
apparent when examining its cytotoxic effects (3). The cytotoxic
functions of NO are observed when it is produced in much larger amounts
by macrophages, hepatocytes, and other cells following their exposure
to cytokines or microbial products. Interestingly, NO produced via such
high output systems inhibits the proliferation of intracellular
pathogens and tumor cells. These effects can be explained by the
reactivity of NO with iron in the Fe-S centers of important
macromolecules, including aconitase and complexes I and II of the
electron transport chain (4-6). The high affinity of NO for iron
probably results in both the removal of iron from [Fe-S] centers and
the formation of dinitrosyl iron species within [Fe-S] proteins (for
review see Ref. 7). NO has been shown to form complexes with a variety
of important iron-containing proteins such as ferritin (8),
ribonucleotide reductase (9), heme-containing proteins (10-12), and
ferrochelatase (13). In fact, ferritin has been suggested to act as a
store of NO (8), and NO-mediated iron release from ferritin has been
shown in vitro (14).
Interestingly, co-cultivation of tumor cells with activated macrophages
results in the inhibition of target cell DNA synthesis and a
concomitant loss of a large fraction (64% per 24 h) of
intracellular iron (15). It was speculated that the loss of iron may be
due to the NO-mediated release of iron from enzymes such as
mitochondrial aconitase (4, 16). In contrast to the work of Hibbs and
others (4, 17, 18), it has been suggested that NO targets loosely bound
pools of non-heme iron, rather than mitochondrial Fe-S clusters (19).
Nonetheless, the relationship between NO and iron is clearly demonstrated by the identification of Fe-nitrosyl complexes in activated macrophages and their tumor cell targets (17, 18, 20, 21). In
these investigations, electron paramagnetic resonance spectroscopy
detected signals typical of Fe-dithiol dinitrosyl (Fe(RS)2(NO)2) complexes and heme-nitrosyl
complexes (17-23).
The important relationship between NO and cellular iron metabolism is
underlined by the fact that NO can also increase the RNA binding of
iron-regulatory protein 1 (IRP1), which plays an important role in
regulating intracellular iron homeostasis (for reviews see Refs. 3 and
24). The effect of NO on IRP1-RNA binding activity occurs via two
possible mechanisms, a direct effect on the [4Fe-4S] cluster and iron
mobilization from cells (25-29). In terms of the ability of NO to
increase iron release, we have shown that the NO produced by
S-nitroso-N-acetylpenicillamine (SNAP),
S-nitrosoglutathione (GSNO), and spermine NONOate (SperNO) can mobilize 59Fe from prelabeled cells as effectively or
more effectively than the clinically used iron chelator desferrioxamine
(DFO) (29). However, the mechanism of NO-mediated iron efflux remains
unknown (29). It has been hypothesized that NO could be released from cells as a complex composed of NO, iron, and thiol-containing ligands
such as cysteine or reduced GSH (23, 30, 31).
To investigate the mechanism of NO-mediated iron release, we examined
the energy dependence of this process. Surprisingly, we found that the
metabolizable monosaccharides, D-glucose
(D-Glc) and D-mannose (D-Man), can
potentiate NO-mediated iron efflux from a variety of cell types. In
contrast, monosaccharides that cannot be transported into cells or that
are poorly metabolized (e.g. L-glucose and
D-2-deoxyglucose) have no significant influence. The effect
of D-Glc on potentiating NO-mediated 59Fe
release is not due to any change in cellular viability or an effect of
D-Glc on NO production by the NO generators. However, these
data indicate that the levels of GSH are crucial in terms of
NO-mediated iron release. These results suggest that the metabolism of
D-Glc by the hexose monophosphate shunt (HMPS) and the
maintenance of GSH levels are essential for NO-mediated iron
mobilization. Thus, this study demonstrates that there is an important
link between energy and iron metabolism and the interaction of NO.
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EXPERIMENTAL PROCEDURES |
Cell Treatments and Reagents
The NO generator SNAP was synthesized by established techniques
(32) from the precursor compound N-acetylpenicillamine
(Sigma). L-Buthionine-[SR]-sulfoximine (BSO),
cytochalasin B, dipyridyl, D-arabinose,
D-allose, D-glucose,
D-2-deoxyglucose, D-fructose, D-galactose, D-mannose, D-psicose,
D-ribose, D-tagatose, D-xylose, L-glucose, GSH, GSNO, sucrose, and transferrin (Tf) were
obtained from Sigma. SperNO was obtained from Molecular Probes (Eugene, OR). Diethyl maleate (DEM) was obtained from Fluka. Minimum essential medium and RPMI 1640 with and without D-Glc were obtained
from Life Technologies, Inc. Desferrioxamine (DFO) was obtained from Novartis Pharmaceutical Co., Basel, Switzerland. Pyridoxal
isonicotinoyl hydrazone (PIH) and its analogue
2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) were
synthesized by standard techniques (33). All other chemicals were of
analytical reagent quality. The NO generators and other compounds were
dissolved in media immediately prior to an experiment. In studies
lasting more than 4 h with a NO generator, the media were replaced
with fresh solutions to maintain NO levels (29, 34).
Cell Culture
The mouse LMTK
fibroblast cell line was obtained
from the European Collection of Cell Cultures (Salisbury, Wiltshire,
UK). The human SK-N-MC neuroepithelioma cell line and murine J774
macrophage lines were obtained from the American Type Culture
Collection (Manassas, VA). The human HepG2 hepatoma cell line was
kindly provided by Dr. Greg Anderson (Queensland Institute of Medical Research, Brisbane, Australia).
The LMTK
and SK-N-MC cell lines were grown in Eagle's
modified minimum essential medium containing 10% fetal calf serum (CSL Ltd., Melbourne, Australia), 1% (v/v) nonessential amino acids (Life
Technologies, Inc.), 1 mM sodium pyruvate (Life
Technologies, Inc.), 2 mM L-glutamine, 100 µg/ml streptomycin (Life Technologies, Inc.), 100 units/ml penicillin
(Life Technologies, Inc.), and 0.28 µg/ml fungizone (Bristol-Myers
Squibb Co.). The J774 and HepG2 cell lines were maintained in RPMI 1640 containing D-Glc with the same additions as described above
for minimum essential medium. Cells were grown in an incubator (Forma
Scientific) at 37 °C in a humidified atmosphere of 5%
CO2, 95% air and subcultured as described previously (35).
Cellular growth and viability were monitored using phase-contrast
microscopy, cell adherence to the culture substratum, and trypan blue staining.
In experiments examining the effects of monosaccharides, disaccharides,
or pyruvate on 59Fe release from cells, RPMI medium free of
D-Glc and pyruvate was used, and the sugars and pyruvate
were added at the concentration required.
Nitrite Determination
The accumulation of nitrite in cell culture supernatants is
commonly used as a relative measure of NO production (25, 29, 34).
Nitrite was assayed using the Griess reagent that gives a
characteristic spectral peak at 550 nm (36).
ATP Assay
Cellular ATP levels were measured as described by the Sigma ATP
assay kit (catalog number 366) with some modifications. In brief, two
75-cm2 culture flasks containing confluent monolayers of
LMTK
fibroblasts were used for all measurements. After
the appropriate incubation with the metabolic inhibitors (cyanide,
azide, or rotenone) and/or D-Glc, the cells were removed by
incubation in 1 mM EDTA/Ca-Mg-free phosphate-buffered
saline, collected, and then counted using a Neubauer counting chamber.
After this, the cells were homogenized in 0.3 ml of phosphate-buffered
saline and 0.3 ml of 12% trichloroacetic acid by vigorous pipetting.
This solution was then placed on ice for 5 min, and the supernatant was
separated from the pellet by centrifugation at 3000 rpm/10 min/4 °C.
The assay was continued as described by the kit.
Glutathione Assay
GSH was measured using the GSH assay kit from Calbiochem. The
only modifications to the protocol were that 10-fold more cells (2.4 × 107 cells/assay) were used, and the cells were
disrupted using two rounds of freeze-thawing before homogenization.
These modifications markedly improved the sensitivity of the technique.
Protein Preparation and Labeling
Apotransferrin was labeled with 59Fe (Dupont NEN) or
56Fe to produce Fe2-transferrin (Tf) using
standard procedures (35). In all studies, fully saturated diferric Tf
was used. Unbound 59Fe or 56Fe was removed by
exhaustive vacuum dialysis against 0.15 M NaCl adjusted to
pH 7.4 with 1.4% NaHCO3 (35).
Efflux Assay of 59Fe from Cells
Efflux Assay, General Protocol--
Standard techniques were
used to examine the effect of NO and other agents on the efflux of
59Fe from prelabeled cells (29, 33). Briefly, cells were
labeled with 59Fe-Tf (0.75 µM) for 2 h
at 37 °C in D-Glc-containing RPMI. After this
incubation, the cell culture dishes were placed on a tray of ice, the
medium aspirated, and the cell monolayer washed four times with
ice-cold balanced salt solution. The cells were subsequently preincubated for 30 min at 37 °C in the presence or absence of the
appropriate sugar, and then a final efflux incubation (2 h/37 °C)
was performed in the presence or absence of the sugar and/or test
reagents. After this incubation, the overlying supernatant (efflux
medium) was transferred to
-counting tubes. The cells were removed
from the Petri dishes after adding 1 ml of balanced salt solution and
by using a plastic spatula to detach them. Radioactivity was measured
in both the cell pellet and supernatant using a
-scintillation counter (LKB Wallace 1282 Compugamma, Finland). In some experiments the
efflux medium was passed through a 5-kDa molecular mass
exclusion filter (Vivaspin 500, Sartorius AG, Germany) to examine the
molecular weight of the 59Fe released.
Iron Efflux Assay, Effect of Metabolic
Inhibitors--
Established procedures were used to examine the effect
of metabolic inhibitors on 59Fe mobilization (37, 38).
Briefly, cells were labeled for 3 h at 37 °C with
59Fe-Tf (0.75 µM) and washed as described
above. The cells were then preincubated for 30 min at 37 °C with
rotenone (50 µM), cyanide (5 mM), or azide
(30 mM). This medium was subsequently removed, and the
cells were incubated for 3 h at 37 °C with medium containing these agents. After this, the overlying supernatant and cells were
collected in separate tubes as described above. Experiments with
metabolic inhibitors were performed either in the presence or absence
of D-Glc.
Iron Efflux Assay, Effect of Agents That Deplete Reduced
Glutathione--
We examined the effect on 59Fe efflux of
two agents that deplete GSH, namely BSO and DEM (39, 40). The cells
were incubated for 20 h at 37 °C with BSO (0.01 mM), washed twice, and then labeled with
59Fe-Tf (0.75 µM) for 2 h at 37 °C.
The cells were then washed 4 times and reincubated for 2 h at
37 °C in the presence and absence of GSNO (0.5 mM).
After this, the overlying supernatant and cells were separated and
placed in
-counting tubes. To reconstitute GSH levels, cells treated
with BSO were incubated at 37 °C with N-acetylcysteine
(NAC; 1 mM/20 h) (40, 41), washed twice, and then labeled
with 59Fe-Tf (0.75 µM) for 2 h at
37 °C. The cells were subsequently washed 4 times and reincubated
for 2 h at 37 °C with GSNO (0.5 mM) in the presence
or absence of D-Glc. The overlying supernatant and cells
were separated and transferred to
-counting tubes.
In contrast to BSO, a 20-h incubation with DEM (1 mM) was
cytotoxic; therefore, cells were incubated with DEM (1 mM)
for 1 h at 37 °C. By using these conditions, no toxic effects
were observed. All other experimental conditions were similar to those
described for BSO above.
Experimental data were compared using Student's t test.
Results were considered statistically significant when
p < 0.05.
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RESULTS |
The Effect of Monosaccharides and Disaccharides on NO-mediated Iron
Mobilization from Cells Prelabeled with
59Fe-Transferrin--
In initial experiments the effect of
D-Glc was examined on NO-mediated 59Fe release
from prelabeled LMTK
fibroblasts. These cells were used
as our primary model because the effect of NO on their iron metabolism
has been well characterized in our laboratory (29, 42). Prelabeling of
cells with 59Fe-Tf for 2 h followed by reincubation
with media without D-Glc resulted in the release of
1.5 ± 0.1% (3 determinations) of total cellular
59Fe, whereas the addition of D-Glc to control
medium had no effect on 59Fe mobilization (Fig.
1). Reincubation of prelabeled cells with GSNO in the absence of D-Glc increased 59Fe
efflux to 5.6 ± 0.2% (3 determinations), and this increased to
16.5 ± 1.4% (3 determinations) when D-Glc was added
with GSNO (Fig. 1). This experiment was repeated seven times with a
similar result in each case. Statistical analysis of the combined data from seven experiments demonstrated that the addition of
D-Glc to GSNO significantly increased (p < 0.00001) 59Fe release compared with GSNO alone. There was
no change in cellular viability (>97%) in the presence or absence of
D-Glc and/or the NO generators, as determined by trypan
blue exclusion, cell adhesion to the culture plate, and overall
cellular morphology. The 59Fe released from the cells in
the presence of NO was found to be less than 5 kDa, as determined by
Vivaspin 500 size exclusion filters.

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Fig. 1.
Reincubation of 59Fe-labeled
cells with the metabolizable sugars D-glucose and
D-mannose markedly increases 59Fe mobilization
from labeled cells but only in the presence of the NO generator,
GSNO. LMTK fibroblasts were labeled with
59Fe-transferrin (0.75 µM) for 2 h at
37 °C, washed, and then preincubated for 30 min at 37 °C in the
presence or absence of D-glucose, D-mannose, or
D-fructose (11 mM). The medium was then
removed, and the cells were reincubated for 2 h at 37 °C in the
presence or absence of the monosaccharides (11 mM) and GSNO
(0.5 mM). After this, the overlying medium (efflux) and
cells were collected in separate tubes. Results are mean ± S.D.
of 3 determinations of a typical experiment of 7 performed.
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Similar results to those reported above for GSNO were also observed for
the NO generators SNAP and SperNO, suggesting that the effect was not
due to the nature of the NO generator added to cells. The relevant
control compounds for GSNO, SNAP, or SperNO, namely GSH,
N-acetylpenicillamine, or spermine, respectively, had no
effect on 59Fe release from cells as shown in our previous
study (29). Moreover, addition of D-Glc to the control
compounds also had no effect on 59Fe mobilization.
Addition of D-Man to GSNO also enhanced 59Fe
release from cells over that seen with GSNO alone, whereas
D-fructose (D-Fuc) had no effect (Fig. 1).
Statistical analysis of the results from seven experiments showed that
the addition of D-Man to GSNO significantly increased
(p < 0.0006) 59Fe release compared with
GSNO alone. It is relevant to note that D-Man can be
metabolized by fibroblasts (43, 44), whereas D-Fuc is
poorly metabolized (45). To determine whether the effects observed with
D-Glc and D-Man were specific to these
monosaccharides, a wide variety of different aldohexoses, aldopentoses,
ketohexoses, and disaccharides were examined for their ability to
enhance NO-mediated 59Fe mobilization. However, from the
range of sugars examined only D-Glc and D-Man
were effective. Other monosaccharides (e.g.
L-glucose, D-2-deoxyglucose,
D-tagatose, D-allose,
D-Glc-6-phosphate, etc.) or disaccharides (lactose and
sucrose) that are not effectively metabolized or transported across the
cell membrane by fibroblasts (45-50) had no effect on cellular
59Fe mobilization (Fig. 1). These results suggested that
transport of the monosaccharide into the cell and its subsequent
metabolism were essential to enhance NO-mediated 59Fe mobilization.
To test this latter hypothesis further, cells of the hepatocyte lineage
(HepG2 hepatoma) were examined to assess if D-Fuc could
enhance NO-mediated 59Fe release. In contrast to
fibroblasts, hepatocytes can readily metabolize D-Fuc (45),
and using the HepG2 cell line this monosaccharide acted similarly to
D-Glc, increasing NO-mediated 59Fe release to a
level 3-fold greater than that observed in its absence (data not
shown). This experiment again indicated that metabolism of the
monosaccharide was essential for increasing NO-mediated
59Fe release. Further studies using fibroblasts examined
the effect of cytochalasin B that is a potent inhibitor of
D-Glc transport into cells (51, 52). In these studies,
cytochalasin B (50 µM) effectively prevented the
D-Glc-enhanced increase in 59Fe release from
fibroblasts in the presence of GSNO (Fig.
2), again suggesting that transport of
D-Glc into the cell was an essential component of its
stimulatory effect.

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Fig. 2.
An inhibitor of D-Glc transport
into cells (cytochalasin B) markedly prevents the effect of this
monosaccharide at stimulating NO-mediated 59Fe efflux from
labeled cells. LMTK fibroblasts were labeled as
described in Fig. 1, washed, and then preincubated for 30 min at
37 °C with medium containing D-Glc (11 mM)
in the presence or absence of cytochalasin B (50 µM). The
medium was then removed, and the cells were reincubated for 2 h at
37 °C with D-Glc (11 mM) in the presence or
absence of cytochalasin B (50 µM) and the NO generator,
GSNO (0.5 mM). After this, the overlying medium (efflux)
and cells were collected in separate tubes. Results are mean ± S.D. of 3 determinations in a typical experiment of 2 performed.
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The effect of D-Glc to increase NO-mediated
59Fe release from mouse LMTK
fibroblasts was
also seen in a wide variety of cell types including human HepG2
hepatoma cells, human SK-N-MC neuroblastoma cells, and the mouse J774
macrophage cell line (data not shown). These results suggest that the
effect of D-Glc on cellular 59Fe release is not
unique to mouse LMTK
fibroblasts.
Additional experiments examined the effect of D-Glc
concentration on NO-mediated 59Fe release (Fig.
3A). The mobilization of
59Fe increased as a function of D-Glc
concentration and then plateaued at 5 mM which is the
physiological concentration of this monosaccharide in serum (53). A
similar dose-response curve was also found with the metabolizable
monosaccharide D-Man. The D-Glc-stimulated increase in NO-mediated 59Fe mobilization from cells was
observed over reincubation times of 2-6 h (Fig. 3B). In
contrast, there was no significant difference in 59Fe
release from control cells in the presence or absence of
D-Glc (Fig. 3B). To understand further the
mechanism of the NO-mediated 59Fe mobilization, the ability
of D-Glc to increase NO-mediated 59Fe release
was assessed after incubations with 59Fe-Tf from 15 min to
24 h (Fig. 3C). After all labeling periods with
59Fe-Tf, D-Glc added with GSNO stimulated
59Fe release over that seen with GSNO added alone (Fig.
3C). However, as observed with strong iron chelators (54),
the greatest increase in 59Fe release from cells was seen
after the shortest labeling period with 59Fe-Tf
(i.e. 15 min; Fig. 3C). Hence, in the present
studies, NO appeared to act like an iron chelator in terms of its
ability to bind and release intracellular iron. Furthermore, since a
much greater proportion of iron taken up from Tf is in a transit form within cells after shorter rather than longer labeling times (54), it
can be suggested that NO is binding iron from a labile pool rather than
that stored in ferritin.

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Fig. 3.
A, increasing concentrations of
D-Glc potentiate NO-mediated 59Fe release from
labeled cells. LMTK fibroblasts were labeled as described
in Fig. 1, washed, and then reincubated with various concentrations of
D-Glc (0.1-11 mM) in the presence or absence
of the NO generator GSNO (0.5 mM). B,
D-Glc stimulates 59Fe release in the presence
of GSNO but not control medium for reincubation periods of 2-6 h.
Cells were labeled as described in Fig. 1, washed, and then reincubated
for 0.5-6 h at 37 °C in the presence and absence of
D-Glc (11 mM) and/or GSNO (0.5 mM).
C, D-Glc-stimulated 59Fe release in
the presence of NO is greatest after short labeling periods with
59Fe-transferrin. Cells were labeled with
59Fe-transferrin (0.75 µM) for 15 min, 3 h, or 24 h at 37 °C, washed, and then incubated for 30 min in
the presence or absence of D-Glc (11 mM). This
medium was removed and the cells then reincubated for 2 h at
37 °C in the presence and absence of D-Glc and/or GSNO.
After this, the overlying medium (efflux) and cells were collected in
separate tubes. Results in A and B or
C are means ± S.D. of 3 determinations in a typical
experiment of 2 performed.
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Effect of Temperature and Metabolic Inhibitors on NO-mediated Iron
Efflux--
To characterize further NO-mediated iron efflux, the
effect of temperature was investigated. As found previously for a
number of iron chelators (38), NO-mediated 59Fe release was
temperature-dependent. After a 3-h label at 37 °C with
59Fe-Tf (0.75 µM) followed by washing and a
3-h reincubation at 37 °C with SNAP (0.5 mM) or the iron
chelator pyridoxal isonicotinoyl hydrazone (PIH; 0.5 mM),
these agents increased 59Fe release from 4 ± 1% in
control cells to 24 ± 2 and 42 ± 2% (three
determinations), respectively. In contrast at 4 °C, SNAP or PIH did
not increase 59Fe release over that observed for control
cells, which released 6% of cellular 59Fe (data not shown).
To determine whether NO-mediated 59Fe efflux was dependent
on metabolic energy, the effects of three metabolic inhibitors (azide, cyanide, and rotenone) were examined on 59Fe mobilization
(Fig. 4, A and B).
As a relevant control to ensure ATP depletion, the efflux of the
chelator PIH was also examined after incubation with the inhibitors.
Previous studies have shown that depletion of intracellular ATP levels
using rotenone, cyanide, azide, and other inhibitors results in a
marked decrease in PIH-59Fe efflux which occurs by an
energy-dependent mechanism (37, 38). Furthermore, we also
assessed ATP levels in cells after exposure to the metabolic inhibitors
in the presence or absence of D-Glc (Fig. 4C).
In this investigation, cyanide (5 mM), rotenone (50 µM), and azide (30 mM) all decreased PIH- and
NO-mediated 59Fe release from cells in the absence of
D-Glc, the most marked effects being observed after
incubation with rotenone and azide (Fig. 4A). Statistical
analysis of the data from 4 experiments showed that when cells were
incubated with metabolic inhibitors in the absence of D-Glc
there was a significant (p < 0.01) decrease in both
NO- and PIH-mediated 59Fe release and a significant
(p < 0.0001) decrease in ATP levels when compared with
the control.

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Fig. 4.
Metabolic inhibitors prevent NO-mediated
59Fe mobilization from labeled cells in the absence of
D-glucose. LMTK fibroblasts were labeled
for 3 h at 37 °C with 59Fe-transferrin (0.75 µM), washed 4 times, and then preincubated for 30 min at
37 °C with cyanide (5 mM), rotenone (50 µM), or azide (30 mM) in the absence
(A) or presence (B) of D-glucose (11 mM). After this preincubation, the NO generator GSNO (0.5 mM) or the iron chelator PIH (50 µM) was
added in the absence (control) or presence of either cyanide (5 mM), rotenone (50 µM), or azide (30 mM), and the cells were reincubated for 3 h at
37 °C. After this, the overlying medium (efflux) and cells were
collected in separate tubes. C, to examine ATP levels, cells
incubated as described above in the absence or presence of
D-glucose and the metabolic inhibitors cyanide, azide, or
rotenone, were harvested and assayed for ATP levels as described under
"Experimental Procedures." Results are means ± S.D. (3 determinations) in a typical experiment of 4 performed.
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Addition of D-Glc to cyanide and azide largely prevented
their inhibitory effects on NO- and PIH-mediated 59Fe
mobilization (compare Fig. 4, A and B) and
reconstitutes cellular ATP levels (Fig. 4C). In contrast,
D-Glc does not prevent the effect of rotenone at inhibiting
NO-mediated 59Fe release (compare Fig. 4, A and
B), even though D-Glc prevented the inhibitory
effect of rotenone on PIH-mediated iron mobilization (Fig.
4B) and helped to reconstitute cellular ATP levels (Fig. 4C). These data suggest that the mechanism of action of
rotenone at inhibiting NO-mediated 59Fe release is
different from that of cyanide and azide. Collectively, these results
suggested that like 59Fe efflux mediated by PIH (37, 38),
metabolic energy was necessary for NO-mediated 59Fe
mobilization from cells.
Effect of D-Glucose on the Mobilization of Iron
Mediated by Strong Iron Chelators--
Considering the observation
that NO appeared to act like an iron chelator to increase
59Fe release (see Ref. 29 and data above), experiments were
performed to assess whether the increase in 59Fe efflux in
the presence of D-Glc could also be observed with other
strong iron chelators (Fig. 5).
Interestingly, D-Glc only stimulated 59Fe
release in the presence of NO, the addition of D-Glc having no effect on 59Fe release mediated by PIH or dipyridyl
(Fig. 5). Similarly, D-Glc had no effect on the ability of
DFO (0.5 mM) to increase 59Fe release from
labeled LMTK
or SK-N-MC neuroblastoma cells (data not
shown). Together, these results suggested that the effect of
D-Glc on increasing 59Fe mobilization is
relatively specific for NO and was not an effect observed with iron
chelators in general.

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Fig. 5.
D-Glc increases 59Fe
release from prelabeled cells in the presence of the NO generator GSNO
but not strong iron chelators. LMTK cells were
labeled as described in Fig. 1, washed, and then preincubated for 30 min at 37 °C in medium with and without D-Glc (11 mM). After this, the medium was removed and the cells then
reincubated for 2 h at 37 °C in the presence and absence of
D-Glc (11 mM) and GSNO (0.5 mM),
PIH (50 µM), or dipyridyl (0.5 mM). After
this, the overlying medium (efflux) and cells were collected in
separate tubes. Results are means ± S.D. of 3 determinations of a
typical experiment of 3 performed.
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The Effect of D-Glucose on the Rate of Nitrite
Production by NO-generating Agents--
It was possible that the
stimulatory effect of D-Glc on NO-mediated 59Fe
mobilization from cells was due to the ability of this agent to
increase NO release from the NO generators as a function of time. To
ensure that this was not the case, the NO generators were incubated
with D-Glc in the presence and absence of cells, and the
production of nitrite was determined as a function of time (Fig.
6). Nitrite was assayed as it is the
direct result of NO oxidation and has been widely used to assay NO
production by NO generators (55, 56). In each case, the addition of
D-Glc to the NO generators had no effect on nitrite
production (Fig. 6). Nitrite production by GSNO and SNAP increased
linearly as a function of time, whereas nitrite generated by SperNO
increased up to 15 min and then plateaued in the absence of cells or
slightly decreased in the presence of cells (Fig. 6). The latter slight decrease in nitrite levels after incubation of cells with SperNO was
reproducible over 3 separate experiments. Previous studies have shown
that the mechanism of NO release from SperNO is different from the
other NO generators examined (57).

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Fig. 6.
D-Glucose does not affect nitrite
production by the NO generators, SNAP, GSNO, or SperNO in the presence
or absence of cells. The NO generator (0.5 mM) was
incubated in the presence or absence of LMTK fibroblasts
for 5-180 min at 37 °C, and the production of nitrite was measured
spectrophotometrically using the Greiss reagent. Results are means ± S.D. (3 determinations) in a typical experiment of 5 performed.
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The Effect of Pyruvate, Citrate, and Reduced Glutathione on
NO-mediated Iron Mobilization--
D-Glc is metabolized in
cells by two main pathways, via the glycolysis/citric acid cycle or by
the hexose monophosphate shunt (HMPS). As the
D-Glc-mediated increase in NO-mediated iron mobilization from cells could be due to the metabolism of this monosaccharide by
either pathway, the effect on iron mobilization of intermediates from
both was examined. To assess if the citric acid cycle may be involved,
cells were incubated with pyruvate or citrate (1 or 11 mM)
(Fig. 7). Both of these latter substrates
are metabolized by the tricarboxylic acid cycle and can be efficiently
transported into cells and metabolized (58-62). In contrast to
D-Glc that markedly increased 59Fe release,
pyruvate or citrate had no effect (Fig. 7).

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Fig. 7.
Citrate and pyruvate do not increase
NO-mediated 59Fe efflux from cells. LMTK
fibroblasts were labeled with 59Fe as described in Fig. 1,
washed, and then reincubated with D-glucose (11 mM), citrate (1 or 11 mM), or pyruvate (1 or 11 mM) in the presence and absence of the NO generator GSNO
(0.5 mM). After this, the overlying medium (efflux) and
cells were collected in separate tubes. Results are means ± S.D.
of 3 determinations of a typical experiment of 2 performed.
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When D-Glc is metabolized by the HMPS, there is an increase
in the level of NADPH and subsequently the ratio of GSH over GSSG increases (41). Considering this, the change in intracellular redox
state may have a critical effect on intracellular iron metabolism. We
hypothesized that the metabolism of D-Glc by the HMPS
results in an elevation of GSH levels that may affect the redox state of the cell and/or the access of NO to chelatable 59Fe
pools. Indeed, previous studies have shown that NO depletes intracellular GSH and then activates the HMPS (63). Similarly, in the
present experiments, incubation with NO decreased cellular GSH levels
in the absence of D-Glc, whereas when D-Glc was
added to the NO generator it markedly prevented GSH depletion (Fig. 8). Statistical analysis of the results
from 3 experiments demonstrated that NO caused a significant decrease
(p < 0.0001) in GSH levels in the absence of
D-Glc. Hence, these data suggest that D-Glc acts as a metabolic substrate to largely prevent GSH depletion in the
presence of NO.

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Fig. 8.
NO depletes intracellular reduced GSH, an
effect that can be rescued by D-glucose.
LMTK fibroblasts were labeled with
56Fe-transferrin (0.75 µM) for 2 h at
37 °C, washed 4 times, and then preincubated for 30 min at 37 °C
in the presence or absence of D-Glc (11 mM).
The preincubation media were removed, and the cells were then
reincubated for 2 h at 37 °C with media containing either
D-Glc (11 mM) and/or GSNO (0.5 mM).
This medium was then removed, the cells washed, and cellular GSH
measured as described under "Experimental Procedures." Results are
mean ± S.D. of 3 determinations of a typical experiment of 3 performed.
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Considering that the level of GSH could be a critical factor in
NO-mediated iron mobilization, experiments were designed to manipulate
the levels of intracellular GSH and assess its effect on
59Fe mobilization. Cells were depleted of GSH using BSO
which is a highly selective and potent inhibitor of the enzyme
-glutamylcysteine synthetase that is involved in GSH synthesis (39)
(Fig. 9, A-C). Preincubation
of cells with BSO (0.01 mM) alone had no appreciable effect
on 59Fe release compared with control cells incubated with
medium alone, whereas BSO markedly inhibited NO-mediated
59Fe efflux (Fig. 9A). In the same experiment,
to reconstitute cellular GSH levels after exposure to BSO, cells were
then incubated with NAC (1 mM) for 20 h, and the
effect on NO-mediated 59Fe release was examined. Treatment
of BSO-treated cells with NAC slightly increased 59Fe
release compared with the control, whereas NAC totally reconstituted NO-mediated 59Fe release (compare Fig. 9, A and
B). Measurement of cellular GSH concentrations demonstrated
that incubation with BSO reduced GSH levels to less than 40% that seen
with the untreated control (Fig. 9C). In addition, when NAC
was added to BSO-treated cells, it reconstituted GSH to a comparable
level of that observed in cells exposed to NAC alone (Fig.
9C). Statistical analysis of the results from 4 experiments
showed that BSO significantly (p < 0.00001) reduced
GSH levels compared with the control, and treatment of BSO-treated
cells with NAC significantly (p < 0.00001) increased GSH.

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Fig. 9.
The GSH synthesis inhibitor BSO inhibits
NO-mediated 59Fe mobilization, an effect that can be
reversed by incubation of cells with NAC that reconstitutes GSH
levels. A and B, LMTK
fibroblasts were incubated for 20 h at 37 °C with BSO (0.01 mM) and washed twice. To reconstitute GSH levels, cells
treated with BSO were incubated at 37 °C with NAC (1 mM/20 h). The cells were then labeled with
59Fe-transferrin (0.75 µM) for 2 h at
37 °C, washed, and then incubated for 2 h with GSNO (0.5 mM) in the presence of D-glucose (11 mM). After this, the overlying medium (efflux) and cells
were collected in separate tubes. C, LMTK
cells were incubated with BSO (0.01 mM/20 h) or control
medium as described above, then washed twice, and reincubated with NAC
(1 mM/20 h) to restore GSH levels. Cellular GSH was
measured as described under "Experimental Procedures." Results are
means ± S.D. of 3 determinations of a typical experiment of 4 performed.
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To confirm the results with BSO, experiments were performed by
incubating cells for 1 h at 37 °C with the oxidizing agent DEM
(1 mM) which is effective at decreasing cellular GSH levels (40, 64, 65). Treatment with DEM resulted in very similar results to
those reported with BSO above, significantly (p < 0.0001) decreasing GSH levels and inhibiting NO-mediated
59Fe release in 3 separate experiments (data not shown).
Collectively, the results with BSO and DEM indicated that GSH is
essential for NO-mediated 59Fe mobilization from cells.
In good agreement with the results performed with chelators in the
presence or absence of D-Glc (see Fig. 5), a 20-h
incubation with BSO markedly decreased 59Fe release after
exposure to GSNO, viz. from 18.2 ± 0.5 to 4.6 ± 1.6% (3 determinations) (Fig. 10). In
contrast, BSO had no effect on 59Fe mobilization mediated
by DFO (0.5 mM), dipyridyl (0.5 mM), PIH (50 µM), or 311 (50 µM) (Fig. 10). These
results indicate that the involvement of GSH in 59Fe
release was an effect specific for NO rather than iron chelators in
general.

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Fig. 10.
The GSH synthesis inhibitor BSO prevents
NO-mediated 59Fe mobilization from labeled cells but has no
effect on 59Fe release mediated by strong iron
chelators. LMTK cells were incubated for 20 h
at 37 °C with BSO (0.01 mM), washed twice, and then
labeled with 59Fe-transferrin (0.75 µM) for
2 h at 37 °C. The cells were then reincubated for 2 h in
the presence of medium containing D-glucose (11 mM) with or without either GSNO (0.5 mM), DFO
(0.5 mM), PIH (50 µM), 311 (50 µM), or dipyridyl (0.5 mM). After this, the
overlying medium (efflux) and cells were collected in separate tubes.
Results are means ± S.D. (3 determinations) in a typical
experiment of 2 performed.
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DISCUSSION |
This is the first study to demonstrate that NO-mediated iron
mobilization from cells can be markedly influenced by the metabolism of
D-Glc. The results are important because they demonstrate a unique relationship between glucose and iron metabolism. This link has
previously been speculated to exist based on the finding that IRP1 acts
as an RNA-binding protein in the absence of iron and also has aconitase
activity when sufficient iron is present (24).
Our studies demonstrate that the effect of D-Glc at
stimulating NO-mediated iron mobilization is due to its ability to be transported into the cell and then subsequently metabolized. Mono- and
disaccharides that are not transported though cell membranes or that
are poorly metabolized have no effect (Fig. 1). Moreover, the effect of
D-Glc at increasing NO-mediated iron release was not due to
this monosaccharide acting as an iron chelator, since D-Glc
had no effect on iron mobilization from control cells (Fig. 1 and Fig.
3, A and B). In addition, it is of interest that
D-tagatose and D-Fuc can chelate iron (50) but
did not increase NO-mediated iron release, in contrast to
D-Glc and D-Man (Fig. 1). Again, these results
suggest that the metabolism of the monosaccharide is important. The
effect of D-Glc at stimulating NO-mediated 59Fe
release appears to be due, at least in part, to the presence of reduced
GSH within the cell (Figs. 8 and 9). Previous studies have also shown
that in the absence of D-Glc there is a decrease in reduced
GSH (41). This latter effect is due to depletion of NADPH derived from
the HMPS that is necessary for the conversion of oxidized glutathione
(GSSG) to its reduced counterpart (41).
We have clearly demonstrated that incubation of cells with NO decreased
cellular GSH levels in the absence of D-Glc (Fig. 8). In
contrast, when D-Glc was added with the NO generator it markedly prevented the depletion of GSH (Fig. 8). Our results are
strongly supported by previous studies, which showed that NO rapidly
caused depletion of intracellular GSH due to the formation of
intracellular S-nitrosoglutathione which then results in
activation of the HMPS (63). In the presence of NO and the absence of
D-Glc, the ability of the HMPS to reconstitute cellular GSH
levels via the production of NADPH does not appear efficient (Fig. 8).
Our results add significantly to the observations of Clancy and
associates (63) and have implications for both intracellular signaling via the NO-Fe interaction, intermediary metabolism, and NO-mediated cytotoxicity. For instance, the fact that NO markedly influences intracellular iron metabolism may be due, in part, to changes in GSH
levels. This in turn could have major effects on pathways that rely on
iron-containing enzymes that are vital for energy production
(e.g. mitochondrial aconitase; Ref. 4) and DNA synthesis (e.g. ribonucleotide reductase; Ref. 9). Indeed, it is well known that exposure of tumor cells to NO results in the inhibition of
energy production and DNA synthesis due to the action of NO on these
and other iron-containing proteins (4, 9, 16). This may be important
for understanding the large loss of cellular iron observed from tumor
target cells (64% after 24 h) after exposure to activated
macrophages producing NO (15). For instance, the increase in HMPS
activity mediated by NO stimulates GSH synthesis in tumor cells, which
then supplies GSH which is involved in iron release.
The process whereby GSH can facilitate NO-mediated 59Fe
mobilization could be due to a number of possible mechanisms. For
example, GSH may alter cellular redox state by reducing the levels of
oxidants such as hydrogen peroxide (41) which may change the
intracellular distribution of iron or lead to an increase in the
proportion of Fe(II) that may be preferentially bound by NO. However,
this hypothesis does not appear compelling, because in contrast to NO-mediated iron efflux, iron mobilization by the Fe(II) chelator dipyridyl or other chelators was not affected after incubation with
either D-Glc or BSO (Figs. 5 and 10). Alternatively, the
increase in GSH levels after incubation with D-Glc could
result in GSH acting as a ligand that together with NO would complete
the coordination shell of iron. Such a "mixed iron complex" with
both NO and GSH ligands bound to iron may provide an appropriate
lipophilic balance to allow diffusion through the cell membrane or
transport by an appropriate carrier. Regarding this, it is of
considerable interest that electron paramagnetic resonance spectroscopy
can detect signals typical of Fe-dithiol dinitrosyl complexes
(Fe(RS)2(NO)2) in cells exposed to NO
(17-23).
It is probable that the effect of D-Glc on stimulating
NO-mediated iron mobilization from cells is not just due to its effect on GSH metabolism. Indeed, our experiments have shown that like the
iron chelator PIH, NO-mediated 59Fe release is both
temperature- and energy-dependent, suggesting a membrane
transport mechanism could be involved. Whereas the process that may be
responsible for NO-mediated iron efflux has yet to be identified, it is
of interest that an iron export molecule known as ferroportin 1 has
recently been cloned (66). Whether this transporter can export iron
bound to NO or PIH remains a question that requires further investigation.
In summary, this study has demonstrated that NO-mediated iron
mobilization can be potentiated by D-Glc due to the
transport and metabolism of this monosaccharide. This effect was
observed with a range of NO generators (GSNO, SperNO, and SNAP) and
cell types. Significantly, the increase in NO-mediated iron release is
dependent on the presence of reduced GSH within the cell. Our results
clearly demonstrate that there is a relationship between D-Glc and iron metabolism. This may have important
implications for intracellular signaling via NO and also NO-mediated
cytotoxicity of activated macrophages that is due, in part, to iron
release from tumor target cells.