TOXICOLOGICAL SCIENCES 77, 334 –340 (2004)
DOI: 10.1093/toxsci/kfh024
Advance Access publication January 21, 2004
Different Mechanisms Mediate Uptake of Lead
in a Rat Astroglial Cell Line
Jae Hoon Cheong,* ,† ,‡ Desmond Bannon,* ,† ,1 Luisa Olivi,† Yongbae Kim,* ,§ and Joseph Bressler* ,† ,¶ ,2
*Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, 21205;
†Kennedy-Krieger Institute, Baltimore, Maryland, 21205; ‡School of Pharmacy, Sahmyook University, Seoul, Korea; §Department of
Preventive Medicine, Soonchunhyan University, Chunan City, Korea; and ¶Department of Neurology,
Kennedy Krieger Institute, 707 North Broadway, Baltimore, Maryland 21205
Received July 28, 2003; accepted October 13, 2003
The mechanism by which lead (Pb) enters astrocytes was examined in a rat astroglial cell line in order to characterize specific
pathways for transport. Pb uptake was saturable at pH 5.5 and 7.4,
although quantitative differences existed in the Michaelis-Menten
constants. At pH 7.4, the Vmax and Km were 2700 fmoles/mg
protein/min and 13.4 ␮M, respectively, whereas the Vmax and Km
were 329 fmoles/mg and 8.2 ␮M in the buffer at pH 5.5, respectively. The presence of extracellular iron inhibited uptake in a
buffer at pH 5.5 but not at pH 7.4. Cells treated with the iron
chelator deferoxamine displayed higher levels of the iron transporter divalent metal transporter 1 (DMT1) mRNA and protein,
and consistent with increased DMT1 expression, the treated cells
displayed greater uptake of Pb in the buffer at pH 5.5 but not at
pH 7.4. Alternatively, at pH 7.4, the transport of Pb was blocked
by the anion transporter inhibitor 4,4ⴕ-diisothiocyanatodihydrostilbene-2,2ⴕ-disulfonic acid (DIDS), which bound to cell surface
proteins at concentrations that were similar to those that blocked
Pb uptake. DIDS did not inhibit uptake of Pb in the buffer at pH
5.5. Greater uptake of Pb was observed in a buffer containing
sodium bicarbonate, which was abrogated in the presence of
DIDS. In summary, the astroglial cell line displays two distinct
pH-sensitive transport mechanisms for Pb.
Key Words: lead; anion; astrocytes; divalent metal transporter 1.
Lead (Pb) poisoning remains a problem in our society,
especially in urban areas. Exposure to Pb is associated with
poor cognitive development in children at relatively low levels
that previously were thought to be safe (Canfield et al., 2003).
The mechanism underlying the effects of Pb on the brain is
unclear but appears complex, involving different neurotransmitters and different brain regions (Cory-Slechta, 1995). Although many studies have addressed mechanisms by which Pb
1
Present address: U.S. Army, Aberdeen Proving Ground, MD 21010.
To whom correspondence should be addressed at Department of Neurology, Kennedy Krieger Institute, 707 North Broadway, Baltimore, MD 21205.
Fax: (443) 923-2695. E-mail: [email protected].
2
Toxicological Sciences vol. 77 no. 2 © Society of Toxicology 2004; all rights
reserved.
interferes with neural processes, the disposition, especially
cellular uptake, of Pb in the brain has been addressed sparingly.
We do not how Pb crosses the blood– brain barrier, nor do we
understand the mechanism by which Pb is taken up by cells in
the brain. Very early work in rats fed 210Pb demonstrated that
Pb accumulates in astrocytes (Thomas et al., 1973). In cell
culture experiments, a glioma cell line was found to accumulate more Pb than a neuroblastoma cell line (Lindahl et al.,
1999), again suggesting that astrocytes accumulate more Pb
than neurons. In consideration that astrocytes are repositories
for Pb in the brain, factors regulating transport of Pb into
astrocytes would influence the amount of Pb interacting with
neurons. Additionally, several functions performed by astrocytes, including secretion of growth factors (Muller et al.,
1995) or transporting neurotransmitters (Schousboe, 2003),
might be impeded by Pb and contribute to the effects of Pb on
cognition.
Even though toxic metals such as Pb serve no nutritional
requirement, transporters for Pb have been identified. Saturable
transport by voltage-dependent calcium channels has been
shown for Pb in adrenal chromaffin cells (Simons and Pocock,
1987) and by anion exchangers in erythrocytes (Simons,
1986a,b). The H ⫹-driven divalent metal transporter 1 (DMT1)
was shown to mediate the uptake of Pb when expressed in high
levels in Xenopus oocytes (Gunshin et al., 1997), yeast, and
human fibroblasts (Bannon et al., 2002). Calcium channels
have been suggested to mediate the uptake of Pb in a glial cell
line (Kerper and Hinkle, 1997; Legare et al., 1998), but kinetics were not reported. Without establishing saturation and
determining the K m and V max, it is difficult to conclude whether
a transporter is involved in uptake.
Because Pb tends to accumulate in astrocytes, the transport
mechanism for Pb was investigated. We used an astroglial cell
line as a model for astrocytes and observed two transport
mechanisms for Pb that were distinguishable by pH of the
transport buffer and sensitivity to inhibitors. One mechanism
displayed properties similar to DMT1, and the other displayed
properties similar to an anion-dependent transporter.
334
335
UPTAKE OF LEAD IN ASTROGLIAL CELLS
MATERIALS AND METHODS
Materials. Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose
(Mediatech Cellgro); fetal bovine serum (Invitrogen); ␣- 32P-dCTP (Amersham); 55Fe, 54Mn, 65Zn, and 35S-methionine (New England Nuclear); Nitran
paper from Schleicher and Schuell; RNAeasy™ kit from Boehringer Mannheim; random priming kit from Qiagen; RNAasin from Promega; glutathione
beads and protein-A sepharose from Pharmacia; the pMal-c2 cloning vector
and amylose resin from New England Biolabs. BCA reagent was purchased
from Pierce. All other chemicals were of reagent grade and obtained from
Sigma.
Cell culture. A rat clonal astroglial cell line, which was a gift from Drs.
Hossain and Laterra, Kennedy-Krieger Institute, Baltimore, MD, was used as
a model for studying astrocytes. The cell line expresses glial proteins such as
myelin-associated glycoprotein precursor protein and sodium- and chloridedependent glycine transporter 1 (Bouton et al., 2001). The cell line also
expresses glial fibrillary acidic protein, which is expressed by astrocytes, on
western blots (data not shown). Cells were grown in media containing Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose and 10% fetal bovine
serum. Cells were routinely plated in 100-mm dishes and dislodged from the
dishes with 0.25% trypsin in Hank’s balance salt solution.
Northern analysis. Total RNA was isolated using Rneasy according to the
manufacturer’s instructions, and northern analysis was carried out with modifications to a previous procedure (Bannon, 2003; Kim et al., 2000). A 20-␮g
fraction of each sample was denatured with glyoxal/dimethylsulfoxide, subjected to electrophoresis through a 1.0% agarose gel, and transferred directly
to nylon membranes in 3 M NaCl/0.3 M sodium citrate. The RNA fixed to the
membranes was hybridized sequentially with cDNA probes for full length
DMT1 cDNA (from M. Garrick, SUNY at Buffalo) and glyceraldehyde
phosphate dehydrogenase (GAPDH). Probes were labeled with ␣- 32P-dCTP by
random priming, and hybridization was carried out for 18 h at 42°C. Membranes were washed once for 5 min and twice for 60 min in 1⫻ SSC/0.1% SDS
and exposed to high-performance chemiluminescence film.
Uptake assay for Pb. To measure uptake of Pb, cells were plated in
100-mm dishes and used for assays at 3 to 4 days after plating when they were
confluent. Uptake buffer consisted of 140 mM NaCl, 5 mM KCl, 1 mM
Na 2HPO 4, 1 mM CaCl 2, and 0.09 % glucose. For pH assays, 10 mM HEPES
(pH 7.4) or 10 mM of 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.5)
was added to the buffer, and the pH adjusted with HCl or NaOH. The uptake
buffer did not appear to affect cell viability as determined by visual inspection.
Prior to uptake assays, medium was removed and cells washed three times with
uptake buffer. Aliquots of a 1 mM 1:5 Pb:citrate (Pb(NO3) 2:sodium citrate)
solution were added to cells at 37°C (total uptake) and 4°C (nonspecific
uptake) in uptake buffer, followed by incubation for 60 min. Citrate ions
maintain the solubility of Pb in solution (Simons, 1986b). To terminate uptake
and remove nonspecifically bound Pb, cells were placed on ice and incubated
with ice-cold wash buffer (10 mM HEPES, 1 mM EDTA, and 150 mM NaCl)
for 5 min on ice. This procedure was repeated three times. Pb was measured
by lysing cells in matrix modifier solution consisting of 0.2% HNO 3, 0.2 %
ammonium dihydrogen orthophosphate, and 0.1% Triton-X 100 followed by
graphite furnace atomic absorption spectrometry as previously described (Bannon et al., 1994) and modified for cell culture (Bannon, 2003). Standards are
run under the same conditions prior to each analysis. Protein was measured by
BCA protein assay. Uptake was computed by subtracting lead measurements
at 4°C from lead measurements at 37°C. Data are reported in moles/mg
protein/min.
The conditions of the uptake assay, that is washing with a buffer containing
EDTA, and subtracting uptake at 4°C from uptake at 37°, were conducted to
minimize the contribution of Pb binding to cell membranes so that the measurements would reflect Pb taken up by the cells. We reasoned that if the
uptake assays were measuring Pb bound to cell membrane, Pb would leach off
when the cells were returned to growth media. To determine whether Pb was
bound to membranes, an uptake assay was conducted with 10 ␮M lead acetate
at pH 7.4. Instead of lysing the cells after washing, cells were incubated with
growth media for different lengths of time before measuring lead. Interestingly, the amount of cellular lead was constant for the entire length of the
experiment (data not shown), which was 8 h, indicating that the assays measure
lead uptake.
Proteins bound to DIDS. Astroglial cells were grown in 35-mm wells for
4 days and incubated during the final 24 h with 20 ␮Ci/ml of 35S-methionine
in DMEM containing 1/10 the amount of methionine. Cells were treated with
different concentrations of DIDS in phosphate buffered saline for 20 min and
then washed with phosphate buffered saline. Whole cell extracts were prepared
by scraping and lysing cells in a buffer consisting of 10 mM Tris–HCl, pH 7.4,
0.5% Triton-X 100, 1 mM EDTA, and 1 mg/ml each of leupeptin and
apropotin. The concentration of radioactivity in each sample was adjusted to
equal levels with lysis buffer. To immunoprecipitate DIDS-binding proteins,
antisera against a KLH–DIDS conjugate (Garcia and Lodish, 1989) (gift from
Dr. Ana Maria Garcia, Eisai Research Institute) bound to protein A-sepharose
beads was added to extracts that were first treated with protein A-sepharose
beads to eliminate proteins that nonspecifically bind to protein A. The beads
were washed with lysis buffer, suspended in SDS-sample buffer, and heated at
95°C for two min. The beads were centrifuged, and supernatants were subjected to SDS–PAGE, and radioactive proteins were visualized by autoradiography.
Antibody against DMT1. A glutathione-transferase-Nramp2 (DMT1) fusion protein was expressed in a pGEX expression vector (Gruenheid et al.,
1999) (a gift from Dr. F. Canone-Hergaux). The vector was constructed by
cloning nucleotides 1–268 from mouse Nramp2 (DMT1) in frame. Overexpression of the DMT1-glutathione transferase fusion protein and purification of
the fusion protein was carried out on glutathione-sepharose beads as described
by the manufacturer (Pharmacia). The protein was boiled in SDS-sample
buffer and the fusion protein was subjected to SDS–PAGE to verify a homogenous band. One hundred micrograms of protein was mixed with Complete
Freund’s adjuvant and injected into New Zealand White rabbits. A 50-␮g boost
was given in incomplete adjuvant at 2, 3, and 7 weeks after the initial
immunization and the rabbits were bled 10 days after the last boost. To affinity
purify the antibody, the DMT1 sequence from the pGEX sequence was cloned
in frame into the EcoR1 and Sal1 site of the pMAL vector (New England
Biolabs) to express a maltose binding protein–DMT1 fusion protein. The
protein was overexpressed and purified with amylose resin according to the
manufacturer’s instructions. The protein was linked to Sepharose 4B with
CNBr, which served as a resin for affinity purifying the antibody against
DMT1 from sera (Harlow and Lane, 1988).
Statistics. All experiments were repeated at least twice. Data is mean ⫾
SE of three replicates. Nonlinear regression was fitted to a Michaelis-Menten
equation using Graphpad Prism威 Version 2. Two-way ANOVA and Tukey’s
test for significance were also carried out also using Graphpad Prism.
RESULTS
Michaelis-Menten Kinetics of Lead Uptake
To test whether a transporter mediates the uptake of lead, we
examined saturable kinetics. Because previous studies reported
that DMT1 mediates Pb uptake, and that DMT1-mediated
metal transport is optimum at acid pH, kinetics were examined
at pH 5.5 and 7.4. Uptake was saturable in buffer at both pH
values. The V max and K m were 2700 fmoles/mg protein/min and
13.4 ␮M at pH 7.4, respectively, whereas the V max and K m were
329 fmoles/mg and 8.2 ␮M at pH 5.5, respectively (Fig. 1).
Inhibition of Pb Uptake at pH 5.5 and 7.4
The quantitative differences in V max (eight-fold) and K m
(two-fold) at pH 5.5 and 7.4 suggest the possibility of two
336
CHEONG ET AL.
FIG. 1. Pb uptake in astroglial cell cultures at pH 5.5 and 7.4. Pb uptake was measured in the astroglial cells that were cultured for 3– 4 days on 100-mm
plates as described in the Materials and Methods. HEPES was used as a buffer at pH 7.4(A), and MES was used as a buffer at pH 5.5 (B). Specific uptake is
shown, which was measured by subtracting total uptake at 37°C from nonspecific uptake at 4°C. Data points, representing the mean ⫾ SE of three replicates,
were analyzed by fitting a Michaelis-Menten equation using non-linear regression
different mechanisms of Pb transport. To qualitatively distinguish these potentially different transport mechanisms, the
sensitivity of Pb uptake to inhibitors was compared at both pH
values. To measure the involvement of DMT1, competition
with iron was examined. Iron did not inhibit uptake of Pb at pH
7.4 (Fig. 2A). In contrast, an approximately 50% decrease in
transport of 12 ␮M Pb and a 65% decrease in transport of 24
␮M Pb was observed in the presence of 250 ␮M ferrous
ammonium sulfate at pH 5.5, indicating that iron competes
with Pb for transport at pH 5.5 (Fig. 2B).
The Effect of Deferoxamine on Uptake of Pb
Inhibition by iron at pH 5.5 but not pH 7.4 suggested the
involvement of DMT1. If DMT1 mediates the transport of Pb,
then an increase in the expression of DMT1 would be expected
to increase uptake of Pb. The iron chelator deferoxamine has
previously been shown to increase levels of DMT1 in PC12
cells (Roth et al., 2002) and fibroblasts (Tchernitchko et al.,
2002). An increase in levels of DMT1 mRNA was observed in
cells treated with 200 ␮M deferoxamine for 16 h (Fig. 3A).
Two splice variants of DMT1 have been found in the rat that
differ in size (Gunshin et al., 1997) and were also observed in
the cell line. Treatment with deferoxamine resulted in an
increase in levels of both species. One species, represented as
the upper band, has an iron response element that conveys
responsiveness to iron deficiency at the level of mRNA
stability, whereas the lower band does not have the iron response element and does not respond to iron status. The increases in both bands suggests that the treatment with deferoxamine is activating a mechanism common to both species,
which might be a response element located 5⬘ upstream from
the transcription site of the DMT1 gene. A possible response
element is the hypoxia response element, which is located in
the 5⬘ noncoding regions of human DMT1 (Lee et al., 1998)
and is activated in cells treated with deferoxamine (Zaman et
al., 1999).
Consistent with increases in mRNA, an increase in DMT1
protein was also observed (Fig. 3B). Two bands at approximately 65 kDa and 100 kDa were observed on western blots
that were detected with an antibody against an amino acid
sequence shared by both species. The two bands might reflect
differences in glycosylation, since there is a putative glycosylation site on the transporter. Also, different tissues display
DMT1 at different sizes. For example, DMT1 displays a mo-
FIG. 2. The effect of iron on the uptake of Pb at pH 5.5 and pH 7.4. Cultures of astroglial cells were assayed for Pb uptake as described in Figure 1. Uptake
of 12 and 24 ␮M Pb was measured at pH 7.4 (A) and pH 5.5 (B) in the presence or absence of iron (250 ␮M ferrous ammonium sulfate) in Pb uptake buffer
with 2.5 mM ascorbic acid. Bars represent the mean ⫾ SE of 3 replicates. Data analyzed by ANOVA. The inhibition by Fe was significant (p ⬍ 0.01), as indicated
by * as determined by Tukey’s post hoc test.
UPTAKE OF LEAD IN ASTROGLIAL CELLS
337
FIG. 3. The effect of deferoxamine on levels of DMT1 mRNA and protein. (A) Total RNA was isolated from astroglial cell cultures that were treated with
200 ␮M deferoxamine for 16 hours or untreated. DMT1 and GAPDH mRNA were measured by Northern analysis from untreated cells (lanes 1 and 2) and cells
treated with deferoxamine (lanes 3 and 4). DMT1 mRNA has two splice variants that differ in mass in the rat. (B) DMT1 protein was measured by western blots
from untreated cells (lanes 1 and 2) and cells treated with 200 ␮M deferoxamine for 48 h (lanes 3 and 4).
lecular mass of 70 –90 kDa in the kidney (Ferguson et al.,
2001) and 80 –90 kDa in the intestine (Moos et al., 2002).
The increases in levels of DMT1 were associated with a two
to three-fold increase in uptake of Pb at pH 5.5 (Table 1). In
contrast, an increase was not evident at pH 7.4.
The Effect of Inhibitors of Anion Transport
The effects of iron and deferoxamine were absent when the
uptake assay was conducted at pH 7.4, suggesting a different
mechanism in the transport of Pb. Previous studies showed that
stilbenes, including DIDS, were potent inhibitors of Pb uptake
in erythrocytes (Lal et al., 1996; Simons, 1986b). Stilbenes
such as DIDS will compete with anions such as Cl – for binding
to anion exchangers by forming a covalent bond with an
external lysine group on cell surface proteins (Muller-Berger et
al., 1995; Schopfer and Salhany, 1995). The effectiveness of
DIDS to inhibit transport of Pb was examined by measuring
transport of 10 ␮M Pb in cells treated with different concentrations of DIDS. As shown in Figure 4, 100 ␮M DIDS
inhibited transport of Pb at pH 7.4 but not at pH 5.5.
DIDS inhibits several types of anion transporters, and it was
possible that DIDS was blocking the monocarboxylate transporters and inhibiting the uptake of a citrate/Pb complex. To
investigate this possibility, the effectiveness of monocarboxylate transporter inhibitor ␣-cyano-4-hydroxycinnamic acid
and other inhibitors of organic anion transporters were examined on the uptake of Pb. Only DIDS was an effective inhibitor
of uptake of Pb (Fig. 5).
Cell Surface Proteins That Bind DIDS
To verify that DIDS binds to proteins on the surface of the
astroglial cells, an antibody directed against DIDS was used to
immunoprecipitate proteins from cells that were treated with
different concentrations of DIDS. Cells were incubated with
35
S-methionine for 24 h to tag proteins for immunoprecipita-
TABLE 1
Effect of Deferoxamine on Uptake of Pb
Treatment
Ph
Pb (␮m)
Uptake a
control
deferoxamine
control
deferoxamine
control
deferoxamine
5.5
5.5
5.5
5.5
7.4
7.4
3
3
6
6
6
6
40 ⫾ 5.5
170 ⫾ 18.0 b
120 ⫾ 10.3
280 ⫾ 24.8 2
700 ⫾ 65.1
800 ⫾ 79.9
The uptake of lead was measured in cells treated for 48 h with 200 ␮M of
deferoxamine and reported as pmoles/mg protein/min.
b
Significantly different from control (0.05⬍p) at the same substrate concentration tested by ANOVA and Tukey’s post hoc test.
a
FIG. 4. The uptake of Pb in cells treated with DIDS. Astroglial cell
cultures were washed with phosphate buffered saline and treated with different
concentrations of DIDS. The uptake of 10 ␮M Pb in buffer at pH 5.5 and pH
7.4 was measured as described in Materials and Methods. Data points represent
the mean ⫾ SE of three replicates.
338
CHEONG ET AL.
FIG. 5. The effect of inhibitors of anion transporters on uptake of Pb in rat
astroglial cells. Cell cultures were treated with 100 ␮M DIDS, 1 mM furosemide (fur), 1 mM probenecid (prob), 2 mM cyano-hydroxycinnamic acid
(CHC), or 200 ␮M niflumic acid (nif) in PBS for 10 min at 37°C. The cells
were washed with uptake buffer at pH 7.4, and uptake of 10 ␮M Pb was
assayed as described. Bars represent the mean ⫾ SE of 3 replicates. Data
analyzed by ANOVA. The inhibition by DIDS was significant (p ⬍ 0.01) as
indicated by * as determined by Tukey’s post hoc test.
tion. Several bands were identified when the immunoprecipitate was subjected to SDS–PAGE and autoradiography (Fig.
6). Bands at approximately 220 kDa, 125 kDa, and 70 kDa
were observed in cells treated with 5 and 25 ␮M DIDS. At 25
␮M DIDS (lane 3), an additional band at approximately 82
kDa was observed. Additional bands within a molecular weight
range of approximately 45–50 kDa were also observed in cells
treated with 100 ␮M DIDS (lane 4). This response was abrogated by preincubating the lysate with DIDS-BSA, which
blocked binding of the antibody to DIDS bound to cell surface
proteins (lane 5).
FIG. 6. DIDS binds to proteins on cell surface of astroglial cells. Cultures
were incubated with 20 ␮Ci/ml 35S-methionine in serum-free DMEM for 24 h,
washed, and treated with 5 (lane 2), 25 (lane 3) and 100 mM DIDS (lane 4),
or remained untreated (lane 1) as described in Materials and Methods. Cell
lysates were prepared and DIDS-binding proteins were immunoprecipitated
with an antibody against a DIDS-KLH conjugate. Additionally, a lysate from
cells treated with 100 mM DIDS was incubated with 1 ␮g/ml DIDS-BSA for
10 min before adding the antibody against DIDS-KLH (lane 5). The antibody/
antigen conjugate was isolated with protein A/sepharose beads and subjected
to SDS-PAGE and autoradiography.
was inhibited by DIDS but not by iron. The effect of DIDS was
likely by binding to an external domain of the transporter,
because DIDS bound to cells surface proteins on the astroglial
cells at concentrations that also inhibited uptake. In previous
Bicarbonate and Transport of Pb
The effect of bicarbonate on the uptake of Pb was examined
because it has been demonstrated that bicarbonate stimulates
Pb uptake, at least in erythrocytes (Simons, 1986b). A 25 mM
concentration of sodium bicarbonate, which approximates that
found in serum, resulted in an approximately two-fold increase
in uptake of Pb at pH 7.4 (Fig. 7). In addition, pretreating cells
with DIDS inhibited the effect of bicarbonate.
DISCUSSION
The objective of this study was to characterize transport of
Pb in astroglial cells. Similar to Pb transport in erythrocytes
(Simons, 1986b), Caco-2 (Bannon, 2003) and adrenal chromaffin cells (Simons and Pocock, 1987), this work demonstrates a
saturable and temperature-dependent mechanism for Pb uptake
in astroglial cells. In addition, there were at least two distinct
transport mechanisms for Pb that was distinguished by the
effect of extracellular pH. At pH 7.4, the transport mechanism
FIG. 7. The effect of bicarbonate on uptake of Pb. The uptake buffer was
made with boiled water to remove CO 2 so that controls contained nominal
concentration of bicarbonate. The uptake of 3 ␮M Pb and treatment with DIDS
was measured at pH 7.4 as described in Materials and Methods. Bars represent
the mean ⫾ SE of 3 replicates. Data analyzed by ANOVA. The stimulation by
bicarbonate was significant (p ⬍ 0.01) as indicated by * in Tukey’s post hoc
test.
339
UPTAKE OF LEAD IN ASTROGLIAL CELLS
studies, DIDS did not block uptake of Pb in Madin-Darby
canine kidney (Bannon et al., 2000) or adrenal chromaffin cells
(Simons and Pocock, 1987), but strongly blocked uptake in
erythrocytes (Bannon et al., 2000). The differences in the
effectiveness of DIDS to block Pb uptake indicate that different
cell types express different transport mechanisms for Pb. The
identity of the transporter that is sensitive to DIDS is unclear.
In erythrocytes, the sensitivity of Pb transport to DIDS suggested the involvement of an anion exchanger, probably AE1.
It is difficult, however, to reconcile how an anion exchanger
mediates the transport of a divalent cation. Anion exchangers
mediate the uptake of monovalent cations, for example the
exchange of LiCO3 – for Cl – (Funder et al., 1978; Romano et
al., 1995), which retains the anion exchanger’s requirement for
electroneutrality. The exchange of PbCO 3 for Cl – would not be
electroneutral. In erythrocytes the involvement of anion exchanger in mediating the transport of Pb may be unique. This
is because the AE1 in erythrocytes, which is also referred to as
Band 3, is approximately 20% of the plasma membrane, is
expressed only by erythrocytes, and is structurally distinct
from other members of the anion exchanger family (Alper et
al., 2002). Rather than an anion exchanger, we suggest that the
sensitivity of uptake of Pb to DIDS is due to the requirement
for an anion. Bicarbonate stimulated the transport of Pb and
might be the required anion. Interestingly, a member of a
family of ZIP metal transporters that transports zinc has been
described in mammalian cells and was stimulated by bicarbonate (Gaither and Eide, 2000). No study has yet identified these
metal transporters in astrocytes.
Our results suggest that DMT1 mediates the uptake of Pb at
pH 5.5 because (1) iron inhibited transport of Pb at pH 5.5,
which is the optimal pH for DMT1-mediated transport of iron;
(2) an increase in transport of Pb at pH 5.5 and an increase in
expression of DMT1 mRNA and protein were observed in cells
treated with deferoxamine; (3) DMT1 was previously reported
to mediate the transport of Pb in yeast and human fibroblasts
(Bannon et al., 2002). Clearly DMT1 is a transporter for iron
and cadmium in the intestine where the pH is acidic (Leazer et
al., 2002; Park et al., 2002). An important question, however,
is whether DMT1 is a cell surface transporter for Pb, or even
for iron, in the brain because DMT1 requires H ⫹ ions and
works optimally at an acidic pH. The pH of the brain, however,
is neutral or near neutral. In a recent review article, the authors
suggested that the concentration of H ⫹ at pH 7.4 is 40 nM and
would be sufficient for DMT1-mediated metal transport at the
cell surface (Garrick et al., 2003). Another factor to consider is
that the pH of the extracellular fluid might not reflect the pH of
the microenvironment of DMT1, which can be modified by
transporters such as H ⫹–ATPase. Recent studies in kidney
reported DMT1 in the apical membranes of distal convoluted
tubules and thick ascending limbs of Henle’s loop (Ferguson et
al., 2001), which were also sites of iron reclamation (Wareing
et al., 2000). Although the fluid in the distal convoluted tubule
is pH 6.6, which is suboptimal for DMT1-mediated transport,
H–ATPAse colocalized with DMT1 and would provide the H ⫹
needed for iron transport (Ferguson et al., 2001). Hence, the
evidence arguing against the involvement of DMT1 in mediating transport of Pb at pH 7.4 in the astroglial cells might
reflect the absence of a microenvironment needed for providing
the H ⫹ that DMT1 requires. In vivo, the microenvironment of
astrocytes would likely be different.
In summary, the data presented here indicates that the astroglial cells express two different transporters for Pb; a transporter working at pH 7.4 that is inhibited by DIDS but not iron
and a transporter at pH 5.5 that is inhibited by iron not DIDS.
The transporter at pH 5.5 appears to be DMT1; the transporter
at pH 7.4 is unknown at this time.
ACKNOWLEDGMENTS
The research was supported by NIH grant PO1 ES08131 and NIEHS Center
Grant 03819. The authors would like to thank Ana Maria Garcia, Senior
Scientist, Eisai Research Institute for the antibody against DIDS.
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