ToxSci Advance Access originally published online on January 21, 2004
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Toxicological Sciences 77, 334-340 (2004)
Copyright © 2004 by the Society of Toxicology
NEUROTOXICOLOGY |
Different Mechanisms Mediate Uptake of Lead in a Rat Astroglial Cell Line
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,1
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* 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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 µM, respectively, whereas the Vmax and Km were 329 fmoles/mg and 8.2 µM 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 4,4'-diisothiocyanatodihydrostilbene-2,2'-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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 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 Km and Vmax, 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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; RNAeasyTM 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 chloride-dependent 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-µg 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 1x 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 Na2HPO4, 1 mM CaCl2, 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% HNO3, 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 µM 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 µCi/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-µg 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® Version 2. Two-way ANOVA and Tukey’s test for significance were also carried out also using Graphpad Prism.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 Vmax and Km were 2700 fmoles/mg protein/min and 13.4 µM at pH 7.4, respectively, whereas the Vmax and Km were 329 fmoles/mg and 8.2 µM at pH 5.5, respectively (Fig. 1
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Inhibition of Pb Uptake at pH 5.5 and 7.4
The quantitative differences in Vmax (eight-fold) and Km (two-fold) at pH 5.5 and 7.4 suggest the possibility of two 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 µM Pb and a 65% decrease in transport of 24 µM Pb was observed in the presence of 250 µM ferrous ammonium sulfate at pH 5.5, indicating that iron competes with Pb for transport at pH 5.5 (Fig. 2B).
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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 µM 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).
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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 molecular 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.
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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 µM Pb in cells treated with different concentrations of DIDS. As shown in Figure 4, 100 µM DIDS inhibited transport of Pb at pH 7.4 but not at pH 5.5.
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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).
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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 35S-methionine for 24 h to tag proteins for immunoprecipitation. 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 µM DIDS. At 25 µM 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 µM 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).
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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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 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 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 PbCO3 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.
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ACKNOWLEDGMENTS |
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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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NOTES |
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1 Present address: U.S. Army, Aberdeen Proving Ground, MD 21010.
2 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: Bressler{at}kennedykrieger.org.
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