Introduction

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Cadmium is an environmental pollutant that causes adverse effects in various organs. Chronic exposure to cadmium in animals and humans results in preferential renal cadmium accumulation, thereby leading to nephrotoxicity (Goering et al., 1995). Although detrimental effects of cadmium have been well documented, the mechanism of cadmium transport, especially in mammalian cells, remains poorly understood. Several animal studies have shown that cadmium intestinal absorption can be affected by dietary components such as iron, zinc, calcium, vitamin D, and phytic acid (Valberg et al., 1976; Omori and Muto, 1977; Washko and Cousins, 1977; Rose and Quarterman, 1984; Foulkes, 1985; Moon, 1994). In in vitro studies, Cd²⁺ has been extensively used as a potent calcium channel blocker (Jacobson and Turner, 1980; Tsien et al., 1987; Lopez et al., 1989) because the ionic radius of Cd²⁺ is close to that of Ca²⁺. However, cellular uptake of Cd²⁺ can be inhibited by calcium channel blockers (Hinkle et al., 1987), suggesting that Cd²⁺ is incorporated into cells at least partly via calcium channels. Other trace elements, such as zinc and copper, also have been reported to inhibit cadmium uptake in hepatocytes (Blazka and Shaikh, 1992) and intestinal cells (Jumarie et al., 1997). Because cadmium is not an essential trace element, transporters for metals such as calcium, zinc, copper, or iron may also be used for cadmium incorporation. However, the process of transport of each metal into mammalian cells has not yet been fully elucidated (Nelson, 1999).

Recently, Gunshin et al. (1997) isolated a divalent metal transporter 1 (DMT1) from rat intestine as a transporter responsible for transferrin-independent iron uptake in the intestine. DMT1 exhibited a broad substrate specificity for divalent metals such as Zn²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ni²⁺, Pb²⁺, and Cd²⁺. Thus, DMT1 is the first and only characterized mammalian metal transporter that can facilitate the cellular uptake of cadmium. However, the actual contribution of DMT1 to cadmium uptake remains unclear.

The most important cellular factor responsible for cadmium resistance in animals is known to be metallothionein (MT). MT is a low-molecular-weight cysteine-rich protein that can be induced by metals, including cadmium, and can attenuate the toxicity of metals by sequestering them (Kägi, 1993). Previously, to investigate the contribution of non-MT factors for cadmium resistance, we established cadmium-resistant cell lines (Yanagiya et al., 1999) from immortalized MT-null embryonic fibroblasts (Kondo et al., 1999) derived from transgenic mice deficient in MT-I and -II, the major isoforms of MT (Michalska and Choo, 1993). The cadmium-resistant MT-null cells exhibited a marked decrease in cadmium uptake and an increase in cadmium release compared with the parental MT-null cells, suggesting that these changes in cadmium transport have conferred resistance to cadmium (Yanagiya et al., 1999). Thus, the characterization of cadmium accumulation in the cadmium-resistant MT-null cells can lead to an improved understanding of mammalian cellular cadmium transport mechanism.

In this study, the changes in the incorporation of various elements into cadmium-resistant cells were determined using a multitracer technique, which permitted measurement of the incorporation of 20 radioactive tracers simultaneously. Interestingly, the incorporation of Mn²⁺ in cadmium-resistant MT-null cells was approximately 10% of that in parental cells. Therefore, we further examined time- and dose-dependent incorporation of Mn²⁺ and competition of Cd²⁺ and Mn²⁺ uptake by each other in these cell lines. The results indicate that the high-affinity transport system for Mn²⁺ was suppressed in cadmium-resistant cells, and that this pathway may be used for the entry of Cd²⁺ into mammalian cells.

	Materials and Methods

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Cell Culture. A clone of cadmium-resistant MT-null cells, Cd-rB5, established from the SV40-transformed embryonic cells of MT-null mice was used (Yanagiya et al., 1999). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Grand Island, NY) with 10% fetal bovine serum under 5% CO₂ at 37°C. Cd-rB5 cells were maintained in the medium containing 10 µM CdCl₂, and were cultured in Cd²⁺-free medium for 10 days before the following assays were performed.

Preparation of a Radioactive Multitracer Solution. The detailed procedure for preparation of the multitracer solution was described previously (Ambe et al., 1995; Enomoto et al., 1996; Hirunuma et al., 1997). Briefly, a plate of silver was irradiated with the 135 MeV/nucleon ¹⁴N beam from the RIKEN Ring Cyclotron (Saitama, Japan). After irradiation, the silver target, which contained various kinds of radioisotopes, was dissolved in HNO₃. The silver was precipitated as AgCl with concentrated HCl, and the AgCl was filtered out completely, leaving multitracer radioisotopes in carrier- and salt-free states. The solution was evaporated to dryness under reduced pressure and was then dissolved in physiological saline. The multitracer solution contained radioactive elements, including Be²⁺, Na⁺, Ca²⁺, Sc³⁺, VO²⁺, VO₂²⁺, Cr³⁺, Mn²⁺, Fe³⁺, Co²⁺, Zn²⁺, Ga³⁺, AsO₃, SeO₃², Rb⁺, Sr²⁺, Y³⁺, ZrO²⁺, TcO₄, Ru³⁺, Ru⁴⁺, and Rh³⁺.

Measurement of Multitracer Incorporation into Cells. Cd-rB5 and parental cells were cultured at a density of 2 × 10⁵ cells per dish in a 6-cm dish for 24 h, and the medium then was replaced with a serum-free medium. After a 30-min preincubation, 0.1 ml of multitracer solution, which contained an ultra-trace amount (less than 1.0 pmol) of each element, was added to the medium (2 ml), and the cells were incubated for 120 min. The medium was removed, and the cells were washed three times with 4 ml of PBS containing 0.05% EDTA. The dish with the washed cells was placed directly on a Ge-detector, and the radioactivities of multitracers were determined. The average counting time for each sample was approximately 15 h. The energy spectra of -ray emitted by multitracers were analyzed by a least-squares fitting program on a PC9821As computer (NEC, Tokyo, Japan), and energy peaks were assigned to each element using a program and database developed at RIKEN (Ambe et al., 1995; Enomoto et al., 1996; Hirunuma et al., 1997). Cellular elemental incorporation was expressed as a percentage of the total amount added in the medium.

Measurement of Uptake and Release of Mn²⁺. Cd-rB5 and parental cells (2 × 10⁵ cells/6-well dish) were preincubated in serum-free medium for 30 min and then exposed to 0.03 µM [⁵⁴Mn]MnCl₂ (DuPont-NEN, Boston, MA) for 0, 15, 30, 60, and 120 min. After three washings with 2 ml of PBS containing 0.05% EDTA, cells were harvested with 1 ml of PBS containing 2% SDS and were transferred to a test tube. The radioactivity of ⁵⁴Mn was measured with an auto well gamma counter (ALOKA, Tokyo, Japan). Similarly, dose-dependent uptake of manganese was determined at 15 min after the addition of 0.01, 0.1, 0.3, 1.0, 3.0, and 10.0 µM [⁵⁴Mn]MnCl₂. For the measurement of manganese efflux, Cd-rB5 and parental cells were treated with 0.03 or 0.3 µM [⁵⁴Mn]MnCl₂ in serum-free medium for 120 min, washed three times with 2 ml of PBS containing 0.05% EDTA, and incubated for 0, 5, 15, 30, 60, and 120 min in a freshly supplemented ⁵⁴Mn-free medium. After rinsing three times with 2 ml of PBS containing 0.05% EDTA, the cells were harvested with 1 ml of PBS containing 2% SDS and were transferred to a test tube. ⁵⁴Mn concentrations remaining in the cells were determined by the radioactivity of ⁵⁴Mn as mentioned above. Protein concentrations in each sample were determined by Lowry's method (Lowry et al., 1951).

Inhibition of Mn²⁺ and Cd²⁺ Uptake. Cells (2 × 10⁵ cells/6-well dish) were preincubated in serum-free media for 30 min and were treated with 0.03 µM [¹⁰⁹Cd]CdCl₂ (Amersham Pharmacia Biotech, Tokyo, Japan) in the presence of 0, 0.03, 0.06, 0.1, and 0.3 µM MnCl₂. After a 15-min incubation, cells were washed three times with 2 ml of PBS containing 0.05% EDTA and were harvested with 1 ml of PBS containing 2% SDS. ¹⁰⁹Cd radioactivity was measured by an ALOKA auto well gamma counter. Similarly, the uptake rate of [⁵⁴Mn]MnCl₂ (0.03 µM) in the presence of 0, 0.03, 0.06, 0.1, and 0.3 µM CdCl₂ was determined. To examine inhibitory effects of other metals on Cd²⁺ or Mn²⁺ uptake, the 15-min incorporation of [¹⁰⁹Cd]CdCl₂ (0.03 µM) or [⁵⁴Mn]MnCl₂ (0.03 µM) was determined in the presence of a 5-fold excess amount of CdCl₂, MnCl₂, ZnCl₂, CoCl₂, NiCl₂, FeSO₄, or CuCl₂. HeLa, PC12, and Caco-2 cells (2 × 10⁵ cells/6-well dish) were treated with 0.03 µM [⁵⁴Mn]MnCl₂ or [¹⁰⁹Cd]CdCl₂ with or without a 5-fold excess amount of CdCl₂ or MnCl₂, respectively, and the 15-min incorporation of ⁵⁴Mn and ¹⁰⁹Cd was determined in the same way as described above.

pH-Dependent Uptake of Cd²⁺ and Mn²⁺. Cd-rB5 and parental cells (2 × 10⁵ cells/6-well dish) were preincubated in serum-free media for 30 min and were exposed to 0.03 µM [¹⁰⁹Cd]CdCl₂ or [⁵⁴Mn]MnCl₂ for 15 min in the pH-adjusted medium. The pH of the medium was adjusted by the addition of 1 N HCl (pH 5.5-6.5) or 7.5% NaHCO₃ solution (pH 6.5-8.0) to DMEM. After washing three times with 2 ml of PBS containing 0.05% EDTA, the cells were harvested with 1 ml of PBS containing 2% SDS and were transferred to a test tube. The radioactivity of ¹⁰⁹Cd or ⁵⁴Mn was measured by an ALOKA auto well gamma counter.

Data Analyses. All experiments were performed in triplicate. Data were expressed as mean ± S.D., and statistical significance was determined by Student's t test. Lineweaver-Burk plots were used to determine values of K_m and V_max of metal uptake. K_i values were derived from Dixon plots.

	Results

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Altered Metal Accumulation in Cd-rB5 Cells. A multitracer technique was used to identify metal(s) that exhibit altered accumulation in cadmium-resistant MT-null cells in which cadmium accumulation was markedly repressed. Figure 1 shows the incorporation of metals in Cd-rB5 and parental cells that were exposed to multitracer solutions for 120 min. Radioactivity from 11 of 20 elements in the multitracer solution (Fig. 1) was detected. Other elements could not be measured due either to their rapid half-lives or to extremely low cellular incorporation. Incorporations of Be²⁺, Sc³⁺, Cr³⁺, Fe³⁺, Zn²⁺, SeO₃², Rb⁺, Y³⁺, and ZrO²⁺ were similar between Cd-rB5 and parental cells. However, the incorporation of Mn²⁺ in Cd-rB5 cells was approximately 10% of that in parental cells. A reduced accumulation of Mn²⁺ in Cd-rB5 cells was also observed when the multitracer solution was dissolved in DMEM with 10% fetal bovine serum (data not shown). Furthermore, the incorporation of Co²⁺ into Cd-rB5 cells was approximately half of that in parental cells (Fig. 1).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Incorporation of various elements into Cd-rB5 and parental cells. Cd-rB5 (solid column) and parental (open column) cells, which were cultured in serum-free media, were exposed to radioactive multitracers for 120 min. After cells were washed with PBS containing 0.05% EDTA, the radioactivities of the elements incorporated into the cells were determined simultaneously by a Ge-detector. The quantification of radioactivity of each element was performed as described in Materials and Methods. The incorporation of 11 of 20 radioactive elements was successfully determined and expressed as a percentage of the amount added in the medium. Mean ± S.D. (n = 3). Asterisks indicate significant differences from parental cells by t test (*P < .05; **P < .01).

Alteration in Mn²⁺ Uptake in Cd-rB5 Cells. Because screening with multitracer solutions suggested a suppressed transport of Mn²⁺ in cadmium-resistant MT-null cells, time- and dose-dependent uptake of Mn²⁺ was determined in Cd-rB5 and parental cells using commercially available [⁵⁴Mn]MnCl₂. As shown in Fig. 2A, the incorporation of Mn²⁺ (0.03 µM) into Cd-rB5 cells was markedly suppressed, whereas the parental cells exhibited a time-dependent accumulation of Mn²⁺. At 120 min after the addition of Mn²⁺ in the medium, Mn²⁺ incorporation into Cd-rB5 cells was approximately 10% of that in parental cells.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Time- and dose-dependent uptake and release of manganese in Cd-rB5 and parental cells. A, Cd-rB5 (closed circles) and parental (open circles) cells were preincubated in serum-free media and exposed to 0.03 µM [54 Mn]MnCl2 for 0, 15, 30, 60, or 120 min. After washing the cells with PBS containing 0.05% EDTA, 54Mn radioactivity in each sample was measured by gamma counter. B, Cd-rB5 (closed circles and triangles) and parental (open circles and triangles) cells were exposed to 0.03 µM (circles) or 0.3 µM (triangles) [54Mn]MnCl2 for 120 min, washed with PBS containing 0.05% EDTA, and then supplemented freshly with 54Mn-free media. The radioactivity of 54Mn remaining in the cells was measured at 0, 15, 30, and 60 min after the medium change. C, Cd-rB5 (closed circles) and parental (open circles) cells were exposed to 0.01, 0.1, 0.3, 1.0, 3.0, and 10.0 µM [54Mn]MnCl2 for 15 min. 54Mn radioactivity in each sample was measured as described above. Mean ± S.D. (n = 3). Asterisks indicate significant differences from parental cells by t test (** P < .01).

Mutual Inhibition of Cd²⁺ and Mn²⁺ Uptake in Parental Cells. To test whether the transport of low concentrations of Mn²⁺ and Cd²⁺ into cells is mediated via the same pathway, we determined whether the uptake of Cd²⁺ and Mn²⁺ is mutually competitively inhibited. As shown in Fig. 3A, Cd²⁺ uptake by parental cells was inhibited by Mn²⁺ in a dose-dependent manner. Similarly, the uptake of Mn²⁺ was also inhibited by Cd²⁺ in a dose-dependent manner (Fig. 3B). However, when Cd-rB5 cells were used, Mn²⁺ did not inhibit Cd²⁺ uptake (Fig. 3A), nor did Cd²⁺ inhibit Mn²⁺ uptake (Fig. 3B). These results suggest that Mn²⁺ and Cd²⁺ share the same process of transport into cells at low concentrations, and that this process is not functioning in Cd-rB5 cells.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Dose-dependent inhibition of cadmium and manganese uptake by each other in parental cells but not in Cd-rB5 cells. A, cells were exposed to 0.03 µM [109Cd]CdCl2 in the presence of 0.03, 0.06, 0.15, or 0.3 µM MnCl2 for 15 min. The radioactivity of 109Cd incorporated into parental (hatched columns) or Cd-rB5 (open columns) cells was measured as described in Materials and Methods. The rates of 109Cd uptake were expressed as relative percentages to those obtained in the absence of MnCl2. Mean ± S.D. (n = 3). Asterisks indicate significant differences from the value obtained in the absence of MnCl2 by t test (*P < .05; **P < .01). B, cells were exposed to 0.03 µM [54Mn]MnCl2 in the presence of 0.03, 0.06, 0.15, or 0.3 µM CdCl2 for 15 min. The measurement of 54Mn incorporation was performed as described in A. Mean ± S.D. (n = 3). Asterisks indicate significant differences from the value obtained in the absence of CdCl2 by t test (**P < .01).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Kinetic profiles of inhibitory effect of manganese on cadmium uptake. A, Lineweaver-Burk plot for the uptake rate of Cd2+ in parental cells. Cells were preincubated in serum-free DMEM for 30 min and exposed to 0.02, 0.03, 0.04, 0.06, or 0.1 µM [109Cd]CdCl2 in the presence (open circles) or absence (closed circles) of 0.3 µM MnCl2 for 15 min. The radioactivity of 109Cd incorporated into the cells was measured as described in Materials and Methods. B, Dixon plot analysis for the inhibition of Cd2+ uptake by MnCl2. Cells were preincubated in serum-free DMEM for 30 min and exposed to 0.02 (open circles), 0.04 (closed circles), or 0.06 µM (open squares) [109Cd]CdCl2 in the presence of various concentrations of MnCl2 for 15 min. The velocity of Cd2+ uptake was measured as described above.

Inhibition of Cd²⁺ and Mn²⁺ Uptake by Other Metals. To investigate the effects of other metal ions on the uptake of Cd²⁺ and Mn²⁺ at low concentrations, the uptake of Cd²⁺ or Mn²⁺ was determined in the presence of a 5-fold excess amount of Zn²⁺, Co²⁺, Ni²⁺, Fe²⁺, and Cu²⁺. As shown in Fig. 5A, Cd²⁺ uptake was inhibited by Zn²⁺ as well as by Mn²⁺, but the other metals did not exhibit inhibitory effects. Similarly, Mn²⁺ uptake was also inhibited by both Cd²⁺ and Zn²⁺, but inhibition by other metals was not observed (Fig. 5B). In Cd-rB5 cells, however, Zn²⁺ did not inhibit either Cd²⁺ or Mn²⁺ uptake (data not shown). These results suggest that Zn²⁺ may also have an affinity for the high-affinity Mn²⁺ and Cd²⁺ transport system. However, Zn²⁺ incorporation was not reduced in Cd-rB5 cells compared with parental cells when Zn²⁺ was added to the medium as a component of multitracer solution (Fig. 1). Thus, Zn²⁺ may not be incorporated into cells solely via the high-affinity Mn²⁺ and Cd²⁺ uptake system.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effects of various metals on the uptake of cadmium or manganese in parental cells. A, parental cells were preincubated in serum-free DMEM for 30 min and exposed to 0.03 µM [109Cd]CdCl2 in the presence of 5-fold excess amounts of MnCl2, ZnCl2, CoCl2, NiCl2, FeSO4, or CuCl2 for 15 min. The rates of 109Cd uptake were expressed as relative percentages to those obtained in the absence of metal inhibitors. B, parental cells were preincubated in serum-free DMEM for 30 min and exposed to 0.03 µM [54Mn]MnCl2 in the presence of 5-fold excess amounts of CdCl2, ZnCl2, CoCl2, NiCl2, FeSO4, or CuCl2 for 15 min. The rates of 54Mn uptake were expressed as relative percentages to those obtained in the absence of metal inhibitors. Mean ± S.D. (n = 3). Asterisks indicate significant difference from the value obtained in the absence of metal inhibitors by t test (**P < .01).

Mutual Inhibition of Cd²⁺ and Mn²⁺ Uptake in Other Mammalian Cells. To test whether the high-affinity transport system for Mn²⁺ and Cd²⁺ is present in other mammalian cells, the influence of low concentrations of Cd²⁺ and Mn²⁺ on uptake of each metal was determined in HeLa, PC12, and Caco-2 cells. As shown in Table 1, the presence of 5-fold excess amounts of Cd²⁺ or Mn²⁺ efficiently inhibited the uptake of 0.03 µM Mn²⁺ or Cd²⁺, respectively, in HeLa, PC12, and Caco-2 cells, although the extents of inhibition differed among cell lines. Thus, the high-affinity Mn²⁺ and Cd²⁺ transport system may exist ubiquitously in mammalian cells.

pH-Dependent Uptake of Cd²⁺ and Mn²⁺. The influence of medium pH on the uptake of Cd²⁺ and Mn²⁺ was determined because the optimal pH of rat DMT1, which can transport both Cd²⁺ and Mn²⁺ as well as Fe²⁺, was reported to be 5.5 (Gunshin et al., 1997). Figure 6 demonstrated that the highest uptake of both Cd²⁺ and Mn²⁺ by parental cells was observed at pH 7.4 to 7.6. These peaks of metal uptake at neutral pH were diminished in Cd-rB5 cells, suggesting that the high-affinity Cd²⁺ and Mn²⁺ transport system functions effectively at physiological pH, and that the transporter that was suppressed in Cd-rB5 cells is not DMT1.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   pH dependence of cadmium and manganese uptake in Cd-rB5 and parental cells. A, Cd-rB5 (closed circles) and parental (open circles) cells were preincubated in serum-free media and exposed to 0.03 µM [109Cd]CdCl2 for 15 min in the medium that was adjusted to specific pH. The radioactivity of 109Cd in each sample was measured as described in Materials and Methods. B, Cd-rB5 (closed circles) and parental (open circles) cells were preincubated in serum-free media and exposed to 0.03 µM [54Mn]MnCl2 for 15 min in the medium that was adjusted to specific pH. Mean ± S.D. (n = 3). The radioactivity of 54Mn in each sample was measured as described in Materials and Methods.

	Discussion

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In this study, we demonstrate that the high-affinity component of the manganese transport system in mammalian cells is also used for cadmium uptake. Previously, we have established cadmium-resistant cell lines from MT-null mouse cells that exhibited a marked decrease in the uptake of cadmium (Yanagiya et al., 1999). The application of multitracer technique in this study revealed that the uptake of ultra-trace amount of manganese in Cd-rB5 cells was reduced to approximately 10% of that in parental cells, whereas no change in the incorporation of zinc, copper, or iron was observed. Because cadmium accumulation in the Cd-rB5 cells was also reduced to 10% of that in parental cells, the same mechanism may be responsible for the reduction of the incorporation of both cadmium and manganese into Cd-rB5 cells.

The results of time- and dose-dependent uptake of Mn²⁺ in Cd-rB5 and parental cells (Fig. 2, A and C) suggest that there are at least two components, having high and low affinity to Mn²⁺, for Mn²⁺ uptake into these cells, and that only the high-affinity component is suppressed in Cd-rB5 cells. However, the low-affinity system(s) for Mn²⁺ transport may not be altered in Cd-rB5 cells nor involved in Cd²⁺ transport. In support of this notion, Cd-rB5 cells did not exhibit cross-resistance to MnCl₂ as determined by cell survival assay (data not shown).

In accordance with our results, several studies have suggested the existence of high- and low-affinity pathways of Mn²⁺ incorporation into cells. In rabbit reticulocytes, a high-affinity (K_m = 0.4 µM) and a low-affinity (K_m = 48 µM) manganese transport system were reported (Chua et al., 1996). Using rat liver slices, Galeotti et al. (1995) reported that there are three saturable systems for Mn²⁺ uptake with different K_m values of 0.075, 2, and 100 µM. Two components of Mn²⁺ uptake were also observed in hepatoma cells (Galeotti et al., 1995). Thus, it appears reasonable that the mouse fibroblast cells used in this study also have both high- and low-affinity pathways of manganese incorporation. To date, however, no specific manganese transporter has been identified.

The apparent K_m values for the uptake of Cd²⁺ and Mn²⁺ at low concentrations were 40 and 36 nM, respectively. The competition study shown in Fig. 3 demonstrated that uptake of low concentrations of Cd²⁺ and Mn²⁺ (0.03 µM) was inhibited by the counterpart metal in parental cells. Kinetic analysis (Fig. 4) revealed that Mn²⁺ inhibited the uptake of Cd²⁺ competitively with a K_i value of 0.14 µM. On the other hand, no inhibition by either metal was observed in Cd-rB5 cells. These data strongly suggest that low concentrations of Cd²⁺ and Mn²⁺ use the same high-affinity pathway for their entry into cells. Furthermore, the absence of the inhibition of Cd²⁺ and Mn²⁺ uptake by one another in Cd-rB5 cells indicates that this pathway is suppressed in Cd-rB5 cells, thereby leading to reduced accumulation of cadmium.

A limited numbers of studies have indicated an interaction between Cd²⁺ and Mn²⁺ in mammalian cell transport systems. Frame and Milanick (1991) demonstrated that the Na⁺-Ca²⁺ exchanger in ferret erythrocytes is able to transport both Mn²⁺ and Cd²⁺ as alternative substrates for Ca²⁺ in Na⁺-free solution. However, the K_m values for uptake of Cd²⁺ and Mn²⁺ in these cells were in a range of 5 to 20 µM, which is much higher than those obtained in this experiment. It has been demonstrated that Cd²⁺ and Mn²⁺ can enter the plasma membrane as surrogates for Ca²⁺ via calcium channels (Shibuya and Douglas, 1992). However, most of these findings were obtained using relatively high concentrations of Cd²⁺ or Mn²⁺ (1-100 µM). Furthermore, the addition of Ca²⁺ in the medium, even at 5 mM, did not inhibit the uptake of 0.03 µM Cd²⁺ or Mn²⁺ in parental cells (data not shown). Thus, it is unlikely that the high-affinity transport system for Mn²⁺ and Cd²⁺ uptake that was observed in this study is mediated via calcium channels.

Recently, the gene for the transferrin-independent iron transporter, DMT1, has been isolated from rat intestine (Gunshin et al., 1997). DMT1 has a broad substrate range, including Zn²⁺, Mn²⁺, and Cd²⁺. Thus, it can be assumed that the transport of Cd²⁺ and Mn²⁺ observed in this study is attributable to DMT1. However, the analysis of pH dependence of Cd²⁺ and Mn²⁺ uptake in Cd-rB5 and parental cells (Fig. 6) revealed that the highest uptake of both Cd²⁺ and Mn²⁺ by parental cells occurred at neutral pH, and the uptake of Cd²⁺ and Mn²⁺ at this range of pH was suppressed in Cd-rB5 cells. On the contrary, DMT1 was reported to be functioning as a proton symporter under acidic conditions like intestinal lumen, and the optimal pH for metal uptake by DMT1 was 5.5 (Gunshin et al., 1997). Furthermore, in the competition experiment (Fig. 5), the addition of other divalent metals such as Fe²⁺, Co²⁺, Cu²⁺, or Ni²⁺ did not inhibit the uptake of Cd²⁺ or Mn²⁺ in parental cells. Thus, a novel transporter that is distinct from DMT1 may be responsible for the high-affinity transport of Mn²⁺ and Cd²⁺ in mouse fibroblast cells. Because the mutual inhibition of the uptake of Mn²⁺ and Cd²⁺ at low concentrations was also observed in other mammalian cells such as HeLa, PC12, and Caco-2 cells (Table 1), the high-affinity Mn²⁺ and Cd²⁺ transport system observed in this study may be present in various mammalian cells.

Several lines of evidence have indicated that cadmium uptake in mammalian cells is mediated at least partly via calcium channels (Jumarie et al., 1997) or the iron transport system (Foulkes, 1985; Moon, 1994). Calcium channels may play a significant role in cadmium transport in excitable cells, and DMT1 can be used for cadmium absorption in the acidic milieu of intestinal lumen. However, little information has been available on the mechanism of cadmium transport in other tissues or cells, and no data have indicated the involvement of manganese in cadmium transport. Due to the existence of both high- and low-affinity Mn²⁺ transport systems, total concentration of manganese in cells or tissues may not exhibit a drastic change when cadmium is applied to cells or administered to animals. This might have led other workers to overlook the involvement of manganese in cadmium transport. Only a few studies have reported that manganese can ameliorate cadmium toxicity (Stacy and Klaassen, 1981; Goering and Klaassen, 1985), although the reduction of cadmium accumulation was not observed in these experiments.

Recent studies in yeast have enabled molecular cloning of multiple metal transporters responsible for the influx of zinc, copper, iron, and manganese (Dancis et al., 1994; Dix et al., 1994; Supek et al., 1996; Zhao and Eide, 1996a,b). However, no specific transporter for metal influx has been isolated in mammals except for DMT1. In this study, establishment of a cadmium-resistant MT-null cell line and the application of multitracer technique have permitted the detection of a novel transport system for both Mn²⁺ and Cd²⁺.