* 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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Key Words: lead; anion; astrocytes; divalent metal transporter 1.
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INTRODUCTION |
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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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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 Dulbeccos modified Eagles 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 Hanks balance salt solution.
Northern analysis.
Total RNA was isolated using Rneasy according to the manufacturers 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 TrisHCl, 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 KLHDIDS 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 SDSPAGE, 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 1268 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 SDSPAGE to verify a homogenous band. One hundred micrograms of protein was mixed with Complete Freunds 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 proteinDMT1 fusion protein. The protein was overexpressed and purified with amylose resin according to the manufacturers 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 Tukeys test for significance were also carried out also using Graphpad Prism.
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RESULTS |
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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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DISCUSSION |
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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 Henles 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, HATPAse 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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NOTES |
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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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