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Acquired Cadmium Resistance in Metallothionein-I/II(–/–) Knockout Cells: Role of the T-Type Calcium Channel Cacn_1G in Cadmium Uptake

Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute and National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (E.M.L., J.L., M.P.W.); and Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas (C.D.K.)

Received May 22, 2005; accepted November 10, 2005

	Abstract

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Metallothioneins (MTs) are cytoplasmic proteins that sequester certain divalent cations and are considered a primary cellular defense against the toxic transition metal cadmium (Cd²⁺). MT-I/II(–/–) knockout [MT(–/–)] cells are available and serve as an excellent tool to study non–MT-related mechanisms in metal tolerance. In the current study, Cd²⁺-resistant MT(–/–) (CdR) and CdR revertant (CdR-rev) cell lines were developed and characterized to investigate non–MT-mediated cellular protection mechanisms. Resistance to Cd²⁺ was approximately 70-fold higher in CdR than the parental MT(–/–) cell line (IC₅₀ = 20 versus 0.3 µM, respectively) and was stable in the absence of Cd²⁺ for 35 days. Accumulation of Cd²⁺ by the CdR cell line was reduced by approximately 95% compared with parental cells, primarily because of a decreased Cd²⁺ uptake. Cd²⁺ uptake by the MT(–/–) parental cell line was independent of sodium, energy, and electrogenic potential. Uptake was saturable (K_m = 65 nM; V_max = 4.9 pmol/mg/min) and pH-dependent (maximal at pH 6.5–7). Potent inhibitors of Cd²⁺ uptake included Zn²⁺ (IC₅₀ = 7 µM), Mn²⁺ (IC₅₀ = 0.4 µM), and the T-type Ca²⁺ channel antagonist mibefradil (IC₅₀ = 5 µM), whereas other metals (including Fe²⁺) and L-type Ca²⁺ channel antagonists had little effect. Immunoblot and real-time reverse transcription-polymerase chain reaction analysis indicated that the Cacn_1G T-type Ca²⁺ channel was expressed at a reduced level in CdR compared with the parental MT(–/–) cell line, suggesting it is important for Cd²⁺ uptake. The CdR1-rev cell line was found to have a Cd²⁺ uptake and sensitivity level in between that of the CdR1 and MT(–/–) cell lines. Consistent with this was an intermediate expression of Cacn_1G in the CdR-rev cell line. These data suggest that decreased expression of Cacn_1G protects cells from Cd²⁺ exposure by limiting Cd²⁺ uptake.

The metallothioneins (MTs) are a family of low-molecular-weight (6–7 kDa), thiol-rich, metal binding proteins (Klaassen et al., 1999). All vertebrates express two or more distinct MT isoforms designated MT I through MT IV (Palmiter, 1998). In mammals, MT I and MT II are ubiquitously expressed in all tissues and are considered the major forms (Palmiter, 1998). Cell lines and transgenic mice deficient in MT are sensitive to Cd²⁺, whereas mice and cells overexpressing MT I and MT II are resistant (Klaassen et al., 1999). In addition, selection of cultured mammalian cells in Cd²⁺ typically results in resistance based on multiple amplifications of the entire MT locus (Palmiter, 1998). MT can bind large amounts of Cd²⁺ and is considered the predominant cellular defense mechanism against Cd²⁺ toxicity (Klaassen et al., 1999).

The level of MT gene expression is impacted by a variety of circumstances and differs widely with factors such as tissue type, cell type, gender, and pathological state (Waalkes and Pérez-Ollé, 2000). In addition, remarkable interindividual variation in MT expression level in human tissues such as liver, kidney, and red blood cells has been reported previously (Onosaka et al., 1985; Bem et al., 1988; Silevis-Smitt et al., 1992; Yoshida et al., 1998; Allan et al., 2000; Wu et al., 2000). Thus, it is important to understand potential non–MT-mediated cellular protection pathways, because these could be important alternative or auxiliary mechanisms for persons who have relatively poor MT expression. Altered cellular transport characteristics could reduce accumulation of Cd²⁺, through either increased efflux or decreased uptake, and could be important for tissue protection. Such mechanisms have not been extensively characterized at the molecular level. A large body of literature exists describing the importance of glutathione-dependent biliary excretion of metals, including Cd²⁺, in vivo (for review, see Leslie et al., 2005). Studies in rats deficient in Mrp2 (Abcc2), an ATP-binding cassette transporter protein involved in the cellular efflux of many xenobiotics, implicate Mrp2 as the major canalicular membrane transporter involved in biliary excretion of Cd²⁺ (Dijkstra et al., 1996; Paulusma and Oude Elferink, 1997). A candidate transporter for cellular uptake of Cd²⁺ is the divalent metal transporter (Dmt1/Slc11a2), also known as the natural resistance-associated macrophage protein 2 or divalent cation transporter 1, a proton-coupled metal-ion transporter (Gunshin et al., 1997). Several in vitro studies have firmly established human DMT1/SLC11A2 as a transporter of Cd²⁺ (Bressler et al., 2004). In addition, a variety of studies have suggested that Cd²⁺ transport into cells occurs through transporters and ion channels involved in the passage of physiological metals such as Zn²⁺, Mn²⁺, and Ca²⁺ (Waalkes and Poirier, 1985; Friedman and Gesek, 1994; Souza et al., 1997; Yanagiya et al., 2000; Zalups and Ahmad, 2003).

Except for the recently identified solute carrier protein ZIP8/Slc39a8 (Dalton et al., 2005), transporters and channels that Cd²⁺ could gain cellular entry through "molecular mimicry" have not been specifically identified at the molecular level. For example, high concentrations of Ca²⁺ channel antagonists have been shown to inhibit cellular uptake of Cd²⁺ (Friedman and Gesek, 1994; Souza et al., 1997). However, concentrations of antagonists used in these studies could inhibit several classes of Ca²⁺ and/or other channel and transport proteins. By using an MT-I/II(–/–) knockout [MT(–/–)] cell line made resistant to Cd²⁺ by continuous exposure in a stepwise manner (Yanagiya et al., 1999), potential non–MT-mediated cellular protection mechanisms from Cd²⁺ have been studied (Yanagiya et al., 1999, 2000; Himeno, 2002; Himeno et al., 2002). The Cd²⁺-resistant MT(–/–) cell line had a reduced ability to take up manganese (Mn²⁺), and it was concluded that the MT(–/–) cell line expresses a novel Mn²⁺ transport system by which Cd²⁺ can enter mammalian cells (Yanagiya et al., 2000). However, such a transporter is yet to be identified.

In the present work, a Cd²⁺-resistant MT(–/–) murine fibroblast cell line (CdR) and a revertant cell line (CdR-rev) have been established and Cd²⁺ transport extensively characterized. Resistance was predominantly because of a reduced Cd²⁺ uptake, probably through a specific decrease in expression of the T-type Ca²⁺ channel Cacn_1G, suggesting that Cacn_1G could be an important pathway for Cd²⁺ entry into cells.

	Materials and Methods

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Materials. [¹⁰⁹Cd]CdCl₂ (3 mCi/mg) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Verapamil, mibefradil, diltiazem, nicardipine, rotenone, sodium azide, choline, MnCl₂ (Mn²⁺), ZnCl₂ (Zn²⁺), NiCl₂ (Ni²⁺), Pb(CH₃COO)₂ (Pb²⁺), FeSO₄ (Fe²⁺), CuCl₂ (Cu²⁺), AlCl₃ (Al³⁺), HgCl₂ (Hg²⁺), NaAsO₂ (As³⁺), and CdCl₂ (Cd²⁺) were purchased from Sigma-Aldrich (St. Louis, MO).

Establishment of Cd²⁺-Resistant and -Revertant Cell Lines. Previously established simian virus 40-transformed MT(–/–) and MT(+/+) fibroblast cells that originated from whole embryonic cells (Kondo et al., 1999) were cultured in high-glucose DMEM with 10% fetal bovine serum under 5% CO₂ at 37°C. CdR cells were developed by continuous exposure to increasing concentrations of Cd²⁺ up to a final concentration of 10 µM, according to the method of Yanagiya et al. (1999). Cells were cultured for several weeks in the presence of 10 µM Cd²⁺ and then cloned by limiting dilution. Cloned cells (CdR1 and CdR2) were grown in the absence of Cd²⁺ (4–10 days) before use in experiments. Revertant cell lines (CdR1-rev and CdR2-rev) were established by growing CdR1 and CdR2 in the absence of Cd²⁺ for up to 238 days.

Cytotoxicity Testing. MT(–/–), MT(+/+), CdR1, CdR2, CdR1-rev, and CdR2-rev cell lines were plated at 1 x 10⁴ cells/well in a 96-well plate, and 24 h later they were treated with Cd²⁺ (0.1–300 µM) for 72 h. The tetrazolium-based microtiter plate assay CellTiter 96 AQ_ueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI), was then used to measure acute cytotoxicity, according to the manufacturer's instructions. Results are expressed as a percentage of untreated control.

Cd²⁺ Total Accumulation, Efflux, and Uptake by MT(–/–), CdR, and CdR1-rev Cells. Cells were plated at 2 x 10⁵ cells/well in six-well plates and cultured 24 h before treatment with carrier-free ¹⁰⁹Cd. For total accumulation studies, cells were incubated with ¹⁰⁹Cd (0.1, 0.3, and 1 µM; 40–400 nCi) for 24 h. Cells were then harvested by washing three times with 2 ml of ice-cold PBS containing 0.05% (w/w) EDTA and lysed with PBS containing 2% SDS. Protein content was measured using the DC Protein Assay kit (Bio-Rad, Hercules, CA), and radioactivity was normalized per milligram of protein. For measurement of cellular Cd²⁺ efflux, cells were incubated for 6 h with ¹⁰⁹Cd (0.1 µM; 40 nCi) in serum-free media, cells were then washed three times with PBS containing 0.05% EDTA (37°C), incubated at 37°C in serum-free media for the indicated time points, and harvested as described above for total accumulation. For uptake studies, cells were incubated with ¹⁰⁹Cd (0.1 µM) in serum-free media for 1 h or at the indicated time points and harvested as described above for total accumulation.

Sodium dependence of uptake was measured using transport buffer (137 mM NaCl replaced with equimolar choline chloride or LiCl, 5.33 mM KCl, 1.8 mM CaCl₂, 0.814 mM MgCl₂, 10 mM HEPES, and 25 mM glucose). The effect of the metabolic inhibitors sodium azide (10 mM) and rotenone (10, 30, and 100 µM) on ¹⁰⁹Cd uptake was assessed in glucose-free DMEM with the addition of mannitol (25 mM) to maintain osmolarity. Cells were preincubated for 1 h with these inhibitors before the addition of ¹⁰⁹Cd and measurement of uptake. The effects of other potential modulators of ¹⁰⁹Cd uptake [Zn²⁺ (0–30 µM), Mn²⁺ (0–30 µM), Ni²⁺ (0–300 µM), Fe²⁺ (0–100 µM), Pb²⁺ (0–100 µM), pH change (pH 5, 6, 6.5, 7, 7.5, or 8), and the Ca²⁺ channel antagonists mibefradil (0–30 µM), verapamil (0–100 µM), diltiazem (0–300 µM), and nicardipine (0–100 µM)] were evaluated, without preincubation of modulators, in serum-free DMEM.

Kinetic parameters of uptake were determined by measuring the initial rate of ¹⁰⁹Cd uptake at eight different substrate concentrations (3–300 nM) at a single time point of 1 h. Kinetic parameters were determined using nonlinear regression analysis (Prism; Graph-Pad Software Inc., San Diego, CA).

Real-Time RT-PCR Analysis. Gene expression was quantified using real-time RT-PCR analysis as described previously (Walker, 2001). In brief, total RNA was isolated from cell lines using an RNeasy midi kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions, reverse transcribed with murine leukemia virus reverse transcriptase and oligo(dT) primers. The forward and reverse primers for selected genes were designed using Primer Express software (Applied Biosystems, Foster City, CA) and are shown in Table 1. The SYBR Green DNA polymerase chain reaction kit (Applied Biosystems) and the MyIQ single-color real-time polymerase chain reaction detection instrument (Bio-Rad) were used for real-time RT-PCR analysis. The relative differences in expression between groups were expressed using cycle time (Ct) values, and the Ct values for genes of interest were first normalized with that of -actin in the same sample, and then differences between groups were expressed relative to controls set as 100%. Assuming that the Ct value is reflective of the starting copy number and there is 100% efficiency, a difference of one cycle is equivalent to a 2-fold difference in starting copy number using the 2^(–dCt) formula. Real-time RT-PCR was performed in triplicate, and similar results were obtained from standard curves produced for each gene analyzed.

Immunoblot Analysis. Cell homogenates were prepared, and relative levels of Cacn_1G were determined by immunoblot analysis essentially as described previously (Yunker et al., 2003). In brief, cells were washed twice with ice-cold PBS and then collected with a plastic cell scraper in 1 ml of HEPES (50 mM; pH 7.4) buffer containing EGTA (1 mM) and protease inhibitors (Complete cocktail tablets; Roche Diagnostics, Indianapolis, IN). Cell homogenates (50 µg) and a positive control homogenate prepared from murine cerebellum (75 µg) were subjected to SDS-polyacrylamide gel electrophoresis and electrotransferred to a nylon membrane. Blots were blocked in 5% (w/v) skim milk powder for 1 h followed by incubation with the CW53 Cav3.1/Cacn_1G antibody in 3% (w/v) bovine serum albumin overnight at 4°C. After washing, blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit antibody (GE Healthcare, Little Chalfont, Buckinghamshire, UK) followed by application of chemiluminescence blotting substrate (SuperSignal Dura Extended Duration; Pierce Chemical, Rockford, IL).

	Results

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Establishment of a Cd²⁺-Resistant Cell Line. A CdR variant of the murine fibroblast MT(–/–) cell line was established by culturing these cells in gradually increasing concentrations of Cd²⁺ up to 10 µM. After establishment and continuous culture in 10 µM Cd²⁺ for 10 weeks, two clones (CdR1 and CdR2) were produced that had similar resistance levels to Cd²⁺ (IC₅₀ of 20 µM), approximately 70- and 7-fold more resistant than the sensitive MT(–/–) parental cell line (IC₅₀ = 0.3 µM) and the MT(+/+) cell line (IC₅₀ = 3 µM), respectively (Fig. 1). To evaluate the stability of the CdR phenotype, the CdR1 cell line was also grown in the absence of Cd²⁺, and the resistance to Cd²⁺ was evaluated over time (Table 2). Resistance was stable for a minimum of 35 days without Cd²⁺ exposure. At 63 days of culture in the absence of Cd²⁺, resistance had decreased 40% with an IC₅₀ of 9 µM versus 15 µM for cells continuously cultured in the presence of Cd²⁺. After 238 days of culture in the absence of Cd²⁺, resistance had decreased by 96% (IC₅₀ = 0.8 versus 18 µM for absence and presence of Cd²⁺, respectively), and this was considered the revertant cell line.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Cytotoxic effect of Cd2+ on MT(+/+), MT(–/–) parental, CdR1, and CdR2 cell lines. The cell lines MT+/+ (), MT–/– parental (), CdR1 (), and CdR2 () were plated at 1 x 104 cells/well in a 96-well plate, and 24 h later they were treated with Cd2+ for 72 h. A tetrazolium-based microtiter plate assay was used to measure acute cytotoxicity as described under Materials and Methods. Symbols represent means of quadruplicate determinations (±S.D.) in a single experiment, and similar results were obtained in three additional experiments.

Resistance Is Mediated Primarily through Reduced Cd²⁺ Uptake. To discern the mechanism of resistance to Cd²⁺, the total accumulation of ¹⁰⁹Cd over 24 h was measured in MT(–/–) parental, CdR1, and CdR2 cell lines (Fig. 2A). CdR1 and CdR2 cell lines accumulated approximately 95% less Cd²⁺ than the MT(–/–) parental cell line over Cd²⁺ concentrations of 0.1, 0.3, and 1 µM. Further experiments revealed that uptake of Cd²⁺ (0.1 µM; 40 nCi) by CdR1 and CdR2 cell lines was reduced by approximately 58, 70, 85, and 92% compared with the MT(–/–) parental cell line at 15, 30, 60, and 120 min, respectively (Fig. 2B). Over 30 min, efflux of Cd²⁺ from the MT(–/–) parental cell line was not detected; however, approximately 40% of Cd²⁺ was effluxed from CdR1 and CdR2 cell lines at 1 min at which point a plateau was reached (Fig. 2C). Although increased efflux does occur in the CdR clones compared with the sensitive parental MT(–/–) cell line, initial Cd²⁺ accumulation was only approximately 5% in the CdR1 and CdR2 cells compared with the parental cell line because of decreased uptake (Fig. 2C). Despite the lack of MT, once Cd²⁺ had entered the MT(–/–) parental cell line it was accumulated (Fig. 2, A–C). This could be because of the lack of an efflux mechanism for Cd²⁺ in this cell line and/or a non-MT mechanism for intracellular sequestration. Overall, these data suggest that a decreased uptake was the primary mechanism by which CdR1 and CdR2 acquired resistance to Cd²⁺.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Cd2+ total accumulation, uptake, and efflux by MT(–/–) parental and CdR cell lines. MT(–/–) parental (white columns or ), CdR1 (gray columns or ), and CdR2 (black columns or ) cell lines were plated at 2 x 105 cells/well in six-well plates and cultured for 24 h before experiments. A, total accumulation of Cd2+ in cells was measured after incubating cells with 109Cd (1 µM, 400 nCi; 0.3 µM, 120 nCi; and 0.1 µM 40 nCi) for 24 h. (B) Cellular uptake of Cd2+ was measured at the indicated time points by incubating cells with 109Cd (0.1 µM; 40 nCi). C, cellular Cd2+ efflux was measured after preincubation of cells for 6 h with 109Cd (0.1 µM; 40 nCi). Columns and symbols represent means of triplicate determinations (±S.D.) in a single experiment, and similar results were obtained in two additional experiments.

Cd²⁺ Uptake Is Independent of Sodium, Energy, and Electrogenic Potential. Because a large part of the CdR phenotype seemed to be conferred by a decreased uptake of Cd²⁺ into CdR1 and CdR2 cell lines, Cd²⁺ influx was characterized using the MT(–/–) parental cell line. When NaCl was substituted with choline or LiCl in transport buffer, cellular Cd²⁺ uptake was increased or unchanged, respectively, indicating that this is not a Na⁺-dependent process (Fig. 3A). When cells were depolarized with 10-fold higher external K⁺(54 mM) than control (5.4 mM) concentrations, little difference in Cd²⁺ uptake was observed, indicating that this process is not dependent on the electrogenic potential of the cell (Fig. 3B). An energy requirement for uptake was evaluated using the metabolic inhibitors sodium azide (10 mM) and rotenone (10, 30, and 100 µM) (Fig. 3C). These inhibitors had no significant effect on Cd²⁺ uptake.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Effect of sodium substitution, potassium excess, and ATP depletion on Cd2+ uptake. The MT(–/–) parental cell line was plated at 2 x 105 cells/well in a six-well plate and cultured for 24 h before experiments. Cells were incubated with 109Cd (0.1 µM; 40 nCi) for 1 h, washed as described under Materials and Methods, and Cd2+ content was quantified. A, sodium dependence of uptake was measured using transport buffer with equimolar choline chloride or LiCl in place of NaCl. B, potassium concentration of the DMEM was adjusted from 5.4 mM (control) to 54 mM during Cd2+ uptake. C, effect of the metabolic inhibitors sodium azide (10 mM) and rotenone (10, 30, and 100 µM) on 109Cd uptake was assessed in glucose-free DMEM with the addition of mannitol (25 mM) to maintain osmolarity. Columns represent means of triplicate determinations (±S.D.) in a single experiment, and similar results were obtained in at least one additional experiment.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Kinetic analysis and pH dependence of Cd2+ uptake. The MT(–/–) parental and CdR1 cell lines were plated at 2 x 105 cells/well in a six-well plate and cultured 24 h before experiments. A, Cd2+ uptake by the MT(–/–) parental cell line was measured at various Cd2+ concentrations (3–300 nM; 12–120 nCi) for 1 h at 37°C. Kinetic parameters were determined from nonlinear regression analysis of the data (GraphPad Prism). Points represent means of triplicate determinations (±S.D.) in a single experiment. The apparent Km for Cd2+ uptake was 65 ± 0.32 nM, and the Vmax was 4.9 ± 0.1 pmol mg–1 min–1. B, MT(–/–) parental () and CdR1 () cell lines were incubated for 1 h at 37°C with Cd2+ (0.1 µM; 40 nCi) at the indicated pH. Symbols represent means of triplicate determinations (±S.D.) in a single experiment, and similar results were obtained in one additional experiment.

Cd²⁺ Uptake Is Modified by Several Metals. The ability of several transition metals and metalloids to alter the uptake of Cd²⁺ was evaluated. As a preliminary experiment, the effect of a single concentration (10 µM) of Pb²⁺, Cu²⁺, Fe²⁺, Al³⁺, Ni²⁺, Zn²⁺, Mn²⁺, Hg²⁺, and As³⁺ on Cd²⁺ (0.1 µM; 40 nCi) uptake was quantified (Fig. 5A). At this concentration, Fe²⁺, Al³⁺, Ni²⁺, Hg²⁺, and As³⁺ had no effect on Cd²⁺ uptake. Several compounds were selected for further characterization through generation of concentration-response curves and IC₅₀ value determination (Fig. 5, B–F).

View larger version (20K): [in this window] [in a new window]   Fig. 5. Modulation of Cd2+ uptake by metals and metalloids. The MT(–/–) parental cell line was plated at 2 x 105 cells/well in a six-well plate and cultured 24 h before experiments. Cells were treated with 109Cd (0.1 µM; 40 nCi) for 1 h at 37°C in the presence of 10 µM concentrations of the indicated metals/metalloids (A) or indicated concentrations of Zn2+ (B), Mn2+ (C), Fe2+ (D), Pb2+ (E), or Ni2+ (F). Points represent means of triplicate determinations (±S.D.) in a single experiment, and similar results were obtained in at least one additional experiment. **, statistically different from untreated control (P < 0.01) (analysis of variance followed by Newman-Keuls post hoc test).

Cd²⁺ Uptake Is Altered by Several Ca²⁺ Channel Antagonists. A series of Ca²⁺ channel antagonists were tested for their ability to inhibit Cd²⁺ uptake by the parental MT(–/–) cell line. Mibefradil, which has been shown to be a relatively specific inhibitor of T-type Ca²⁺ channels, proved to be a potent inhibitor of Cd²⁺ uptake by the parental MT(–/–) cell line with an IC₅₀ value of 5 µM (Fig. 6A). The phenylalkylamine verapamil, an L-type Ca²⁺ channel antagonist, inhibited Cd²⁺ uptake with an IC₅₀ value of 60 µM (Fig. 6B). Diltiazem and nicardipine are drugs from different chemical classes of L-type Ca²⁺ channel antagonists, the benzothiazepines and dihydropyridines, respectively. Neither diltiazem (IC₅₀ > 300 µM) nor nicardipine (IC₅₀ > 100 µM) was a potent inhibitor of Cd²⁺ uptake (Fig. 6, C and D). The overall pattern of inhibition by these Ca²⁺ channel antagonists indicates that a T-type Ca²⁺ channel is involved in Cd²⁺ uptake.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Modulation of Cd2+ uptake by Ca2+ channel antagonists. The MT(–/–) parental cell line was plated at 2 x 105 cells/well in a six-well plate and cultured 24 h before experiments. Cells were incubated with 109Cd (0.1 µM; 40 nCi) for 1 h at 37°C in the presence of mibefradil (A), verapamil (B), diltiazem (C), or nicardipine (D). Points represent means of triplicate determinations (±S.D.) in a single experiment, and similar results were obtained in at least one additional experiment. **, statistically different from untreated control (P < 0.01) (analysis of variance followed by Newman-Keuls post hoc test).

View larger version (31K): [in this window] [in a new window]   Fig. 7. Functional characterization of the CdR1-rev cell line. A, The CdR1 (), CdR1-rev (), and MT–/– parental () cell lines were plated at 1 x 104 cells/well in a 96-well plate, and 24 h later they were treated with Cd2+ for 72 h. A tetrazolium-based microtiter plate assay was used to measure acute cytotoxicity as described under Materials and Methods. Symbols represent means of quadruplicate determinations (±S.D.) in a single experiment, and similar results were obtained in two additional experiments. B, CdR1 (), CdR1-rev (), and MT–/– parental () cell lines were plated at 2 x 105 cells/well in a six-well plate and incubated 24 h before experiments. Cells were incubated with 109Cd (0.1 µM; 40 nCi) for 6 h at 37°C, and then efflux was measured at the indicated time points (as described under Materials and Methods). Points represent means of triplicate determinations (±S.D.) in a single experiment, and similar results were obtained in one additional experiment. C, MT(–/–) parental (), CdR1-rev (), and CdR1 () were plated as described in B, and uptake of 109Cd (0.1 µM; 40 nCi) was measured for the indicated time points at 37°C. Points represent means of triplicate determinations (±S.D.) in a single experiment, and similar results were obtained in one additional experiment. D, MT(–/–) parental (white columns), CdR1-rev (gray columns), and CdR1 (black columns) were plated as described in B and incubated with 109Cd (0.1 µM; 40 nCi) ± Mn2+ (1 µM), Zn2+ (10 µM), or mibefradil (mbf) (10 µM) for 1 h at 37°C. Columns represent means of triplicate determinations (±S.D.) in a single experiment, and similar results were obtained in one additional experiment. *, statistically different from MT(–/–) (P < 0.01); , statistically different from CdR1-rev (P < 0.01); **, statistically different from untreated cell line control (analysis of variance followed by Newman-Keuls post hoc test).

Expression Levels of the T-Type Ca²⁺ Channel Cacn_1G Suggest Potential Importance for Cd²⁺ Uptake. The relative expression levels of the Ca²⁺ channels Cacn_1G and Cacn₃ were found to be decreased in CdR clones compared with the MT(–/–) parental cell line in preliminary microarray analysis [further genomic analysis will be reported in a separate article (E. M. Leslie, J. Liu, D. M. K. Ducharme, and M. P. Waalkes, manuscript in preparation)]. This prompted further characterization of Cacn_1G and Cacn₃ expression in the CdR1-rev, CdR1, and MT(–/–) parental cell lines using real-time RT-PCR and immunoblot analysis (Fig. 8). Consistent with the intermediate function of the CdR1-rev cell line, the Cacn_1G T-type Ca²⁺ channel was expressed at a level between that of the CdR1 and MT(–/–) parental cell lines. Thus, the Cacn_1G T-type Ca²⁺ channel was expressed in CdR1 cells at 0.2% of the expression in the MT(–/–) parental cell line and at approximately 8% of the CdR1-rev (Fig. 8A). The protein expression of Cacn_1G was also analyzed and confirmed the RNA expression level differences between the cell lines (Fig. 8B). Protein expression was not detectable in cell homogenate prepared from the CdR1 cell line, a faint band was visible in the CdR1-rev, whereas the MT(–/–) parental cell line contained a bright band at a molecular mass of 240 kDa, consistent with the murine cerebellum-positive control. The CW53 antibody had been rigorously tested for Cacn_1G specificity in a previous study (Yunker et al., 2003). The other Ca²⁺ channel subunit, Cacn₃ (Fig. 8C), did not differ in expression levels between the CdR1 and CdR1-rev cell lines, indicating that this protein is unlikely to influence Cd²⁺ uptake.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Real-time RT-PCR and immunoblot analysis of Ca2+ channel expression. A, MT(–/–) parental (white columns), CdR1 (black columns), and CdR1-rev (gray columns) cell lines were grown in the absence of Cd2+ for 4 to 10 days, RNA was isolated, and expression levels of the Ca2+ channel Cacn1G were analyzed using real-time RT-PCR as described under Materials and Methods. Columns represent means of triplicate determinations, and similar results were obtained with RNA isolated from the CdR2 and CdR2-rev cell lines. B, immunoblot of cell (50 µg) and tissue (70 µg) homogenates prepared from CdR1 cell line, murine cerebellum (positive control), MT(–/–) parental, and CdR1-rev cell lines. Homogenates were resolved on a 6% acrylamide gel, transferred, and probed with the polyclonal CW53 as described under Materials and Methods. C, MT(–/–) parental (white columns), CdR1 (black columns), and CdR1-rev (gray columns) cell lines were grown in the absence of Cd2+ for 4 to 10 days, RNA was isolated, and expression levels of the Ca2+ channel Cacn3 were analyzed using real-time RT-PCR as described under Materials and Methods. Columns represent means of triplicate determinations, and similar results were obtained with RNA isolated from the CdR2 and CdR2-rev cell lines. *, statistically different from MT(–/–) uptake (P < 0.05); , statistically different from CdR1-rev uptake (P < 0.05) (analysis of variance followed by Newman-Keuls post hoc test).

Expression Levels of Dmt1/Slc11a2, AE4/Slc39a7, and Zip8/Slc39a8 Are Unchanged. The expression levels of two Mn²⁺ transporters, Dmt1/Slc11a2 and AE4/Slc39a7, were measured because of the proposed importance of Mn²⁺ transporters in Cd²⁺ uptake. Dmt1/Slc11a2 mRNA levels were not significantly different in the MT(–/–) cell line compared with the CdR1-rev or the CdR1 cell lines (Fig. 9A). AE4/Slc39a7 has been proposed to transport Mn²⁺ (Eide, 2004); however, expression was similar in the CdR1 versus MT(–/–) parental cell line (Fig. 9B) and therefore unlikely to be involved in the Cd²⁺ resistance observed. The expression of ZIP8/Slc39a8, a solute carrier protein that has recently been shown to facilitate uptake of Cd²⁺ in transfected cell lines and is thought to be associated with Cd²⁺ sensitivity (Dalton et al., 2005), was similar in the CdR1 versus the MT(–/–) parental cell line (Fig. 9C). Therefore, ZIP8/Slc39a8 is unlikely to be involved in the differential Cd²⁺ uptake and sensitivity in the CdR and MT(–/–) parental cell lines.

View larger version (19K): [in this window] [in a new window]   Fig. 9. Real-time RT-PCR analysis of Dmt1/Slc11a2, AE4/Slc39a7, ZIP8/Slc39a8, Zrtl1/Slc39a4, and aquaporin 1 (Aqp1) expression in MT(–/–) parental, CdR1, and CdR1-rev cell lines. Cell lines were grown in the absence of Cd2+ for 4 to 10 days, RNA was isolated, and expression levels of Dmt1/Slc11a2 (A), AE4/Slc39a7 (B), ZIP8/Slc39a8 (C), Zrtl1/Slc39a4 (D), and aquaporin 1 (E) were analyzed in MT(–/–) parental (white columns), CdR1-rev (gray columns), and/or CdR1 (black columns) cell lines using real-time RT-PCR as described under Materials and Methods. Columns represent means of triplicate determinations, and similar results were obtained with RNA isolated from the CdR2 and CdR2-rev cell lines. *, statistically different from MT(–/–) uptake (P < 0.05); , statistically different from CdR1-rev uptake (P < 0.05) (analysis of variance followed by Newman-Keuls post hoc test).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this work, a Cd²⁺-resistant MT(–/–) cell line was established and characterized to investigate non–MT-mediated cellular protection mechanisms. In addition, a revertant cell line (CdR-rev) was developed and proved to be a powerful tool for the determination of gene expression changes relevant or incidental to development of the Cd²⁺ resistance phenotype. Through functional and molecular analysis of the differences between the revertant, resistant, and parental MT(–/–) cell lines, we have significantly extended previous work (Yanagiya et al., 1999, 2000) by more precisely defining the transport process involved in cellular Cd²⁺ uptake. Genetic, biochemical, and functional analyses suggest that the T-type Ca²⁺ channel, Cacn_1G, is an important pathway for cellular entry of Cd²⁺. Likewise, evidence implies that decreased expression of Cacn_1G is a protective mechanism for cells exposed to Cd²⁺. Decreased Cacn_1G expression may be a key factor in the cellular response to Cd²⁺ exposure and may provide for acquired tolerance in cells that poorly produce MT. Given the frequently observed interindividual variability of MT expression seen in human subjects (Onosaka et al., 1985; Bem et al., 1988; Silevis-Smitt et al., 1992; Yoshida et al., 1998; Allan et al., 2000; Wu et al., 2000), knowledge of secondary factors in acquired Cd²⁺ tolerance could be very important.

Previous studies have suggested that Cd²⁺ can enter cells through L-type Ca²⁺ channels (Friedman and Gesek, 1994; Souza et al., 1997), and in the current study, uptake of Cd²⁺ was inhibited by verapamil, an L-type Ca²⁺ channel antagonist. However, verapamil has also been reported to inhibit T-type Ca²⁺ channels at concentrations as low as 10 µM, whereas more specific inhibition of L-type channels occurs at low micromolar concentrations (Heady et al., 2001). Consistent with the possibility of non–L-type channel inhibition by verapamil was the observation that two other L-type channel antagonists, diltiazem and nicardipine, had no effect on Cd²⁺ uptake. In contrast with verapamil, mibefradil blocks T-type Ca²⁺ channels 10 to 30 times more potently than L-type Ca²⁺ channels (Heady et al., 2001). Electrophysiological experiments using a variety of T-type Ca²⁺ currents have shown mibefradil IC₅₀ values ranging from 0.1 to 4.7 µM (Heady et al., 2001). Thus, the ability of mibefradil to inhibit Cd²⁺ uptake by the MT(–/–) parental cell line with an IC₅₀ of 5 µM is consistent with inhibition of a T-type Ca²⁺ channel. Unlike other members of the T-type Ca²⁺ channel protein family, Cacn_1G, has been shown to be relatively insensitive to Ni²⁺ inhibition (Lacinová et al., 2000). Thus, it has been previously reported that Ni²⁺ inhibits Cacn_1G with an IC₅₀ value of 470 µM, comparable with the inhibitory potency of Ni²⁺ observed in the MT(–/–) parental cell line (IC₅₀ = 300 µM) (Lacinová et al., 2000). Combined with these previous results, the present data implicate T-type Ca²⁺ channels as a critical component of Cd²⁺ entry into cells.

Uptake of Cd²⁺ was not affected by the metabolic inhibitors rotenone or sodium azide, suggesting a passive mechanism, consistent with a channel. T-type Ca²⁺ channels are "activated" or opened by low-voltage currents close to the resting membrane potential of the cell. High extracellular concentrations of K⁺ are often used as a depolarizing stimulus to activate voltage-dependent Ca²⁺ channels and therefore increase Ca²⁺ influx into cells (Jagannathan et al., 2002). Thus, our observation that Cd²⁺ uptake by MT(–/–) parental cells was unchanged by increased K⁺ concentrations is inconsistent with a voltage activation mechanism. However, previous studies have shown that recombinant Cacn_1G channels expressed in human embryonic kidney 293 cells are insensitive to increased K⁺ concentrations, unless cell membranes contain endogenous ion channels, such as Kir2.1, that stabilize resting membrane potential (Kim et al., 2004). Therefore, the uptake of Cd²⁺ in our cell system is possibly not affected by increased K⁺ concentrations, because such endogenous ion channels are absent. Indeed, preliminary real-time RT-PCR analysis of Kir2.1 expression in the MT(–/–) cell line suggested that it is present at very low levels (data not shown).

In vivo, Cacn_1G is expressed in many brain regions with especially high levels in amygdala, thalamus, subthalamic nuclei, and cerebellum (Perez-Reyes et al., 1998; Yunker et al., 2003). In addition, Cacn_1G is expressed at lower levels in heart, placenta, lung, and kidney (Perez-Reyes et al., 1998; Andreasen et al., 2000). In the rat, Cacn_1G mRNA is expressed in all regions of the kidney, including the distal and proximal tubules. Immunohistochemistry of rat kidney showed that Cacn_1G is localized to the apical plasma membrane of distal convoluted tubule, connecting tubule principal cells and inner medullary collecting duct principal cells (Andreasen et al., 2000). Cd²⁺-induced renal injury is dependent upon uptake and accumulation of this metal and although speculative, expression of Cacn_1G in this tissue could have implications for Cd²⁺ toxicity (Friedman and Gesek, 1994).

Dmt1/Slc11a2 is a transporter expressed at the apical surface of cells in many mammalian tissues and at particularly high levels in the small intestine and kidney (Gunshin et al., 1997). Evidence strongly suggests that Dmt1/Slc11a2 plays an important role in the absorption of ferrous iron and potentially other divalent metals in the proximal duodenum (Canonne-Hergaux et al., 1999; Leazer et al., 2002). Although Dmt1/Slc11a2 is a candidate transporter of Cd²⁺, uptake in the MT(–/–) parental cell line indicated that this process was not Dmt1/Slc11a2-mediated. For example, uptake of Cd²⁺ by the MT(–/–) cell line was potently inhibited by Zn²⁺, maximal at slightly acidic to neutral pH, insensitive to Fe²⁺, and mediated with very high affinity. In contrast, transport by Dmt1/Slc11a2 is insensitive to Zn²⁺, inhibited by Fe²⁺, maximal at pH 5.5, and mediated at lower affinity (K_m = 1 µM) (Gunshin et al., 1997; Okubo et al., 2003). Gene expression analysis indicated that there was no difference in the Dmt1/Slc11a2 mRNA expression in MT(–/–) parental versus the CdR or CdR-rev cell lines consistent with the non-Dmt1/Slc11a2 phenotype.

Experiments conducted by Yanagiya et al. (2000) using an independently derived Cd²⁺-resistant MT(–/–) cell line also suggested that a non-Dmt1/Slc11a2 Cd²⁺ uptake mechanism was present in the parental MT(–/–) cell line. Their Cd²⁺-resistant cell line had a reduced ability to take up Mn²⁺, and Mn²⁺ was a potent inhibitor of Cd²⁺ uptake (K_i = 0.14 µM) (Yanagiya et al., 2000). Thus, the authors concluded that the MT(–/–) cell line expresses a novel high-affinity Mn²⁺ transport system through which Cd²⁺ can enter the cell. In the current study, expression of AE4/Slc39a7, a member of the SLC39 family of metal ion transporters, a proposed Mn²⁺ transporter (Eide, 2004), was expressed at similar levels in CdR and MT(–/–) parental cell lines, suggesting it is not involved in the observed alterations in Cd²⁺ uptake. Very little is known about the molecular pathways by which Mn²⁺ enters mammalian cells. Although further experiments are required to be conclusive, there are examples of overlap in Ca²⁺ and Mn²⁺ uptake (Van Baelen et al., 2004); Mn²⁺ has been shown to permeate through a related T-type Ca²⁺ channel, Cacn_1H (Kaku et al., 2003); and potentially a common mechanism exists (i.e., Cacn_1G) for the passage of Cd²⁺, Ca²⁺, and Mn²⁺ into the MT(–/–) cell line.

The aquaporins are a family of membrane proteins that allow rapid transport of water across lipid membranes (Law and Sansom, 2002). Certain aquaporins, the aquaglyceroporins, will also allow the downhill movement of uncharged solutes such as glycerol and urea (Liu et al., 2004). It is noteworthy that several aquaglyceroporins, including human AQP7 and AQP9, have been shown to be important for arsenic passage into cells (Liu et al., 2004). Analysis of CdR1, CdR2, and MT(–/–) parental cell lines indicated that the expression of aquaporin 1 was decreased in the CdR versus the parental cell line and that aquaporin 1 expression was intermediate in the CdR-rev cell line, consistent with the intermediate Cd²⁺ resistance phenotype. The functional implications of the decreased RNA expression of aquaporin 1 are not clear. Unlike the aquaglyceroporins, aquaporin 1 transport has been shown to be highly selective for water molecules (Law and Sansom, 2002). The high-resolution X-ray structure of bovine aquaporin 1 (Sui et al., 2001) indicates that the channel pore is not wide enough to allow the passage of a hydrated ion, such as Cd²⁺, and not energetically favorable for dehydration to occur before entering the channel (Law and Sansom, 2002). Therefore, it is highly unlikely that aquaporin 1 is directly involved in translocation of Cd²⁺ across the plasma membrane, but it could have a different function such as in osmoregulation. Indeed, aquaporin 1 is lost from the brush-border membrane of kidney proximal tubules of rats treated with Cd²⁺, suggesting that this channel is affected by the metal in vivo (Sabolic et al., 2002).

In conclusion, understanding non–MT-mediated cellular protection mechanisms is important because of the interindividual variability in MT expression in humans. In addition, such mechanisms could work in synergy with MT to provide protection from this toxic metal. This work clearly indicates that decreased cellular uptake of Cd²⁺ is a very important mechanism of acquired Cd²⁺ resistance. The thorough characterization of Cd²⁺ transport in MT(–/–) cells has lead to the identification of Cacn_1G as a potential pathway for cellular uptake of Cd²⁺. Although further research is required to understand the physiological relevance of our observations, down-regulation of Cacn_1G expression could be very important for cellular protection from Cd²⁺ under normal circumstances and for persons with MT(–/–) deficiencies.

	Acknowledgements

 
We thank Dr. John S. Lazo (University of Pittsburgh, Pittsburgh, PA) for the kind gifts of the MT(–/–) and MT(+/+) cell lines. We thank Dr. Maureen W. McEnery (Case Western Reserve University, Cleveland, OH) for the generous gifts of the CW53 antibody and murine cerebellum homogenates. We also thank Drs. Larry Keefer, Wei Qiu, and Jean Francois Coppin for critical review of the manuscript.

	Footnotes

 
This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. E.M.L. is the recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship and a Davies Charitable Foundation Research Fellowship Award.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.014241.

ABBREVIATIONS: MT, metallothionein; Dmt, divalent metal transporter; CdR, Cd²⁺-resistant; Cdr-rev, Cd²⁺-resistant revertant; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; Ct, cycle time; Zirtl, zinc-iron regulated transporter-like.

¹ Current affiliation: School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina.

Address correspondence to: Dr. Michael P. Waalkes, Inorganic Carcinogenesis Section, National Cancer Institute, National Institute of Environmental Health Sciences, P.O. Box 12233, Mail Drop F0-09, 111 Alexander Dr., Research Triangle Park, NC 27709. E-mail: waalkes{at}niehs.nih.gov

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