Introduction

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The tripeptide GSH plays numerous critical roles in mammalian cells, including maintenance and regulation of cellular thiol and redox status and serving as a substrate in various xenobiotic metabolism reactions (Reed, 1990). Within renal proximal tubular cells, there are at least two major pools of GSH, one in the cytoplasm and one in the mitochondrial matrix (Schnellmann et al., 1988). Cellular fractionation studies showed that the mitochondrial pool in rabbit and rat renal proximal tubule comprises approximately 15 to 30% of total cellular GSH (Schnellmann et al., 1988; Lash et al., 1998), which equates with the approximate volume fraction of mitochondria in the proximal tubules. GSH is the predominant nonprotein thiol in mitochondria, and its presence in this organelle at millimolar levels is necessary to maintain redox status, and hence activity of numerous sulfhydryl-dependent processes (Beatrice et al., 1984; Yagi and Hatefi, 1984; Lê-Quôc and Lê-Quôc, 1985, 1989). Accordingly, selective depletion of the mitochondrial pool of GSH has been associated with enhanced susceptibility to several types of chemical-induced toxicity, such as oxidative stress (Martensson and Meister, 1989; Reed, 1990; Shan et al., 1993; Fernandez-Checa et al., 1997). Furthermore, decreased concentrations of mitochondrial GSH are associated with S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced acute toxicity (Lash and Anders, 1987) and apoptosis (Chen et al., 2001) in renal mitochondria and renal proximal tubular cells, respectively; apoptosis in general (Kroemer et al., 1998); and chronic ethanol exposure and alcoholic liver disease in liver mitochondria (Fernandez-Checa et al., 1991, 1993, 1997).

The predominant, if not exclusive, localization of enzymes catalyzing de novo synthesis of GSH is the cytoplasm (Griffith and Meister, 1985; Lash et al., 1998), indicating that transport of the intact tripeptide from the cytoplasm across the mitochondrial inner membrane must occur to supply the mitochondria with its pool of GSH. GSH does not diffuse passively across the mitochondrial inner membrane. Rather, because mitochondria possess a membrane potential with the matrix space negative relative to the cytoplasm and because GSH is a negatively charged molecule at physiological pH, GSH must be transported actively or in exchange for another anion (Smith et al., 1996).

Eight known anion carriers are present on the mitochondrial inner membrane that could conceivably play a role in the mitochondrial uptake of GSH from the cytoplasm. In previous studies (McKernan et al., 1991; Chen and Lash, 1998; Chen et al., 2000), we examined the role of these carriers by assessing substrate specificity and patterns of inhibition, and determined that two carriers, the dicarboxylate carrier (DCC) and the oxoglutarate carrier (OGC), are responsible for at least 80% of the total uptake of cytoplasmic GSH into renal cortical mitochondria. These two carriers, along with the mono- and tricarboxylate carriers, share a common 30-kDa molecular mass subunit and are believed to belong to a carrier "superfamily" (Palmieri et al., 1996). Function of each of these carriers is electroneutral, catalyzing the exchange of anions across the inner membrane without any net transfer of charge.

Both the DCC and OGC proteins have been purified to homogeneity and reconstituted into proteoliposomes (Bisaccia et al., 1985, 1988; Kaplan and Pedersen, 1985; Lançar-Benba et al., 1994). Two difficulties with studying the purified DCC protein from mammalian tissues relates to its low abundance in the inner mitochondrial membrane and its instability in its purified form, due to autoxidation and inactivation (Kaplan and Pedersen, 1985; Bisaccia et al., 1988). To circumvent this limitation, the cDNA for the DCC from Norway rat was isolated using related expressed sequence tags homologous to genes in Caenorhabditis elegans and yeast (Kakhniashvili et al., 1997). The rat DCC was then expressed in Escherichia coli and functionally reconstituted into proteoliposomes (Fiermonte et al., 1998).

In this communication, we significantly expand on this earlier report (Fiermonte et al., 1998) and our prior studies of GSH transport in mitochondria from rat kidney proximal tubules (Chen and Lash, 1998; Lash et al., 1998), by expressing recombinant rat DCC (as the N-His₆ fusion protein). By reconstituting purified DCC into proteoliposomes and transiently expressing DCC in a cell line derived from rat kidney proximal tubules (NRK-52E), we show that DCC transports GSH and malonate with properties similar to those observed in intact rat kidney mitochondria. The recombinant DCC-His₆ in transfected NRK-52E cells localizes in mitochondria where it mediates GSH uptake, thereby demonstrating function of the carrier in an intact renal cell. Furthermore, overexpression of the DCC in NRK-52E cells markedly protected against apoptosis induced by either DCVC or tert-butyl hydroperoxide (tBH), suggesting the toxicological significance of the mitochondrial GSH transport process.

	Materials and Methods

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Materials. [³H-glycyl]GSH (44.8 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). [2-¹⁴C]Malonate (56 mCi/mmol) was purchased from ICN Pharmaceuticals (Costa Mesa, CA). -Glutamyl amino acids, S-alkyl GSH derivatives, and ophthalmic acid were purchased from Sigma-Aldrich (St. Louis, MO), Bachem California (Torrance, CA), or Roche Applied Science (Indianapolis, IN). Restriction enzymes for PCR, other enzymes (e.g., DNA polymerases and T7 RNA polymerase), and plasmids were purchased from New England Biolabs (Beverly, MA), Promega (Madison, WI), and Invitrogen (Carlsbad, CA). PCR primers were custom synthesized by Integrated DNA Technology, Inc. (Coralville, IA). Cloning vectors (pGEM-T Easy, pRSET, and pcDNA3.1/V5-His-TOPO) were purchased from Promega and Invitrogen. Dowex-1, Triton X-100, Percoll, and phospholipids were purchased from Sigma-Aldrich. Materials for gel electrophoresis (SDS, acrylamide, agarose, and buffers) were purchased from Bio-Rad (Hercules, CA) or Sigma-Aldrich. ProBond nickel-chelating resin was purchased from Invitrogen. Millicell polycarbonate filter inserts (0.4-µm pore size, 25 mm in diameter) were purchased from Millipore Corporation (Bedford, MA). NRK-52E cells (catalog number CRL-1571), cell culture medium (catalog number 30-2002; Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, and 1.0 mM sodium pyruvate), and 10% (w/v) bovine calf serum were purchased from American Type Culture Collection (Manassas, VA). Antibodies to the His₆ fusion proteins were purchased from Invitrogen. Double-distilled, deionized water was used for all experiments. All other chemicals and reagents were purchased from commercial vendors and were of the highest purity available.

Amplification of Rat Kidney Mitochondrial DCC cDNA by reverse transcription-PCR and Bacterial Expression. Total RNA was previously isolated from renal cortical homogenates of male Fischer-344 rats (200-300 g b.wt.; Charles River Laboratories, Inc., Wilmington, MA) by acid phenol extraction using the TRIzol extraction kit (Invitrogen) and was stored at 80°C until needed. Total rat kidney RNA was reverse transcribed with Superscript II reverse transcriptase and amplified with forward and reverse primers based on the complete cDNA sequence (1946 base pairs) for the mitochondrial DCC protein from the Norway rat (Rattus norvegicus; GenBank accession number AJ223355). PCR primers were 5'-CGG GCC AGG TCG CTG CTG CTC T-3' (sense, positions 8-29) and 5'-GCC AAG GCA GGG TCG GAA GGC GTA G-3' (antisense, positions 1039-1063), where the coding region is from positions 34 to 894; PCR conditions were 30 cycles of 94°C × 3 min for denaturation and 68°C × 2 min for annealing/extension. The 1056-base pair product was ligated into a T-A cloning vector (pGEM-T Easy) for transformation. Confirmation of the PCR product was by automated DNA sequencing.

Preparation of Proteoliposomes for Reconstitution of Purified DCC. Liposomes and proteoliposomes were prepared essentially as described previously (Chen et al., 2000). Briefly, liposomes were prepared by transferring 700 mg of phosphatidylcholine into a 30-ml polypropylene tube and drying under a stream of nitrogen. After redissolving the dried lipid in diethyl ether and removal of solvent under nitrogen, liposome buffer (7 ml; 120 mM HEPES, 50 mM KCl, and 1 mM EDTA, pH 7.0) was added to the dried lipid, the tube was flushed with nitrogen, sealed, and vigorously mixed on a vortex mixer. Lipid was then dispersed in a bath sonicator for approximately 60 min and was stored at 80°C for up to 3 months. Proteoliposomes were prepared by mixing purified carrier protein (200 µl, 50 µg of protein) or liposome buffer (for a blank control) with 0.5 ml of liposomes and 50 µl of substrate solution (buffer containing 20 mM phosphate and 20 mM malate) in a final volume of 1 ml. After mixing on a vortex mixer, the proteoliposomes were rapidly frozen in liquid nitrogen and stored at 80°C for up to 1 month until use. Immediately before assay, the proteoliposome suspension was thawed, sonicated with a bath sonicator at room temperature, placed on ice for 1 min, and then passed through an anion exchange column (Dowex-1, 100-200 mesh, equilibrated with liposome buffer and prepared in a 9-inch Pasteur pipette) to remove external substrate by elution with liposome buffer. The opalescent fraction (approximately 1 ml) contains the proteoliposomes and was collected. The proteoliposomes were equilibrated at room temperature for approximately 5 min before being used in transport assays. This freeze-thaw cycle and sonication method give rise to a heterogeneous population of large, primarily unilamellar proteoliposomes that are suitable for transport studies (Kaplan and Pedersen, 1985).

Transport Assays in Reconstituted Proteoliposomes. Transport of substrates (i.e., GSH and malonate) into proteoliposomes was measured by first preincubating 200 µl of proteoliposomes (preloaded with 1 mM phosphate and 1 mM malate) for 1 min with 12 µl of "reconstitution buffer" (120 mM HEPES, 50 mM KCl, and 1 mM EDTA, pH 7.4). Competitive inhibitors were added simultaneously with substrates. Transport was initiated by addition of 12 µl of 20× radiolabeled substrate. At various times (30-240 s), 30-µl aliquots were transferred to a microcentrifuge tube containing 70 µl of reconstitution buffer + 40 mM of an irreversible transport inhibitor (i.e., pyridoxal 5'-phosphate), and tubes were mixed and placed on ice until the next processing step. After completion of time courses, samples were loaded onto Sephacryl S-100 mini-columns, which were prepared in 5.75-inch Pasteur pipettes. These columns were placed in 16 × 125-mm glass test tubes and were centrifuged at 2300 rpm × 2 min in a tabletop clinical centrifuge. Supernatant from each tube was transferred into a 5-ml scintillation vial, and each tube was washed with 200 µl of scintillation fluid, which was then added to scintillation vials.

Culture of NRK-52E Cells. NRK-52E cells were cultured on collagen-coated, polystyrene T-25 or T-175 culture flasks with Dulbecco's modified Eagle's medium containing 4% (w/v) glutamine, 1.5 g/l sodium bicarbonate, 4.5 g of glucose/l, 1 mM sodium pyruvate, and 10% (v/v) bovine calf serum in an atmosphere of 5% CO₂/95% air at 37°C. On reaching confluence (5-9 days), subcultures were prepared by a 15-min treatment with 0.02% (w/v) EDTA, 0.05% (w/v) trypsin solution and replating the cells at a density of 4 × 10⁴ cells/cm².

Transfection of NRK-52E Cells. NRK-52E cells were transiently transfected with rat kidney DCC cDNA to overexpress mitochondrial GSH transport activity. Plasmid DNA was purified following amplification in E. coli cells, using the Promega Wizard Prep purification kit. cDNA encoding the rat kidney DCC was subcloned into the pcDNA3.1/V5-His-TOPO vector and transfected into the NRK-52E cells by either the calcium phosphate method (Current Protocols in Molecular Biology, 1999) or with FuGENE 6 from Roche Applied Science. For the calcium phosphate method, purified plasmid containing 200 mM CaCl₂ in HEPES buffer, pH 7.4, was added dropwise to subconfluent T-175 flasks to a final concentration of 20 µg/10 ml of Dulbecco's modified Eagle's medium. After 24 h, medium was removed and the cells were washed twice with phosphate-buffered saline (PBS). After 48 h, cells were harvested by incubating with Cellstripper (Mediatech, Herndon, VA) for 5 min and gentle scraping. These harvested cells were then used for isolation of mitochondria for transport measurements and Western blot analyses (see below). For the FuGENE 6 method, transient transfections were carried out as described by the manufacturer using a 3:1 ratio of FuGENE 6 reagent to plasmid DNA. After 24 h, cells were harvested as described above.

Isolation of Mitochondria from NRK-52E Cells and Transport Measurements. Mitochondria were prepared from seven confluent T-175 flasks, which contain approximately 70 to 85 × 10⁶ NRK-52E cells, by differential centrifugation as described previously (McKernan et al., 1991). Yield of mitochondrial protein was 6 to 10.5 mg. Purity of mitochondria was determined by measurement of activities of marker enzymes for mitochondria (succinate dehydrogenase and glutamate dehydrogenase), plasma membrane (alkaline phosphatase, (Na⁺ + K⁺)-stimulated ATPase), cytoplasm (lactate dehydrogenase), and endoplasmic reticulum (glucose 6-phosphatase) in whole cells and the mitochondrial fraction. Most of the total cellular activity of the mitochondrial marker enzymes (75%) was recovered in the mitochondrial fraction, whereas minimal-to-modest amounts of total cellular activity of markers for the other subcellular organelles were recovered in this fraction. Specifically, approximately 5% of total cellular activity of markers for the plasma membrane and endoplasmic reticulum and <2% of total activity of the cytoplasmic marker enzyme were recovered in the mitochondrial fraction. This indicates that the mitochondrial fraction obtained from the NRK-52E cells is of high purity and has minimal contamination with other subcellular organelles.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting. For gel electrophoresis, 10 or 20 µg of protein was loaded per well onto a 10% (w/v) or a 12% (w/v) polyacrylamide gel (Bio-Rad), and separation was achieved according to the method of Laemmli (1970). Total protein was visualized by staining with Coomassie Brilliant Blue G. For Western blot analysis, protein was transferred by electroblotting to a nitrocellulose membrane (MSI, Westborough, MA), blocked with 3% (w/v) bovine serum albumin (Promega), washed with Tris-buffered saline containing Tween 20, probed with a monoclonal anti-RGSHHHH tag antibody (QIAGEN, Valencia, CA), washed with Tris-buffered saline containing Tween 20, and then probed with an anti-mouse IgG antibody conjugated to alkaline phosphatase (Jackson Immunoresearch Laboratories, West Grove, PA). Immunoreactive bands were visualized after incubation with a solution containing 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (Promega).

Flow Cytometry Analysis of Cell Cycle and Quantitation of Apoptosis. Cell cultures were washed twice with sample buffer (PBS plus 1 g of glucose/l filtered through a 0.22-µm filter), dislodged by trypsin/EDTA incubation, centrifuged at 400g × 10 min at 22°C, and resuspended in sample buffer. Cell concentrations were adjusted to 1 to 3 × 10⁶ cells/ml with sample buffer and 1 ml of the cell suspension was centrifuged at 400g × 10 min at 22°C. All of the supernatant except 0.1 ml/10⁶ cells was removed and the remaining cells were mixed on a vortex mixer in the remaining fluid for 10 s. Next, 1 ml of ice-cold ethanol (70%, v/v) was added to the sample in a dropwise manner, with samples being mixed for 10 s between drops. Tubes were capped and fixed in ethanol at 4°C. After fixation, cells were stained with propidium iodide (50 µg/ml) containing RNase A (100 U/ml). Samples were then mixed, centrifuged at 1000g × 5 min at 22°C, and all the ethanol except 0.1 ml was removed. Cells were mixed in the residual ethanol and 1 ml of the propidium iodide staining solution was added to each tube. After mixing again, cells were incubated at room temperature for at least 30 min. Samples were analyzed within 24 h by flow cytometry using an FACSCalibur flow cytometer (BD Biosciences, San Jose, CA), which is a core facility of the National Institute of Environmental Health Sciences Center for Molecular Toxicology with Human Applications at Wayne State University. Analyses were performed with 20,000 events per sample using the ModFit LT version 2 for Macintosh data acquisition software package (Verity Software House, Inc., Topsham, ME; distributed by BD Immunocytochemistry System BDIS). Propidium iodide was detected by the FL-2 photomultiplier tube. Fractions of apoptotic cells were quantified by analysis of the sub-G₁ (subdiploid) peak with ModFit cell cycle analysis. The percentages of distribution of cells in the various stages of the cell cycle (G₀/G₁, S, and G₂/M) were also calculated. Cell aggregates were discarded in the flow cytometry analysis by postfixation aggregate discrimination.

Data Analysis. Data were normalized to protein content, which was determined by the bicinchoninic acid protein assay from Pierce Chemical (Rockford, IL). All measurements were performed three to five times. Significant differences between means were first assessed by a one-way or two-way analysis of variance, depending on the comparisons being tested. When significant "F values" were obtained, the Fisher's protected least significant difference t test was performed to determine which means were significantly different from one another, using a two-tail probability, P < 0.05, as the criteria for significance.

	Results

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Bacterial Expression of the Rat Kidney Mitochondrial DCC. The full-length cDNA for the rat DCC was prepared from rat kidney total RNA by RT-PCR. The cDNA was subcloned into the pRSET-B expression vector for expression of DCC protein as an N-terminal His₆ fusion protein. Expression of DCC in one clone (15) was readily induced with IPTG (Fig. 1A); the arrow shows the time-dependent increase in expression of a 35.5-kDa protein, the expected molecular mass of the DCC-His₆ product. This was confirmed by Western analysis (Fig. 1B). Figure 1A also shows that DCC-His₆ could be solubilized from inclusion bodies. The detergent-solubilized extract from inclusion bodies was fractionated on a Ni²⁺-affinity column. The purified DCC-His₆ was approximately 90% pure by Coomassie staining (Fig. 2). The overall yield of DCC-His₆ from a 1-liter culture of bacteria was typically 20 to 25 mg of protein.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Expression of rat kidney mitochondrial DCC protein in E. coli. Clone 15 of BL21 Lys S cells transformed with pRSET B-DCC cDNA insert was grown for 1, 3, 5, or 22 h in the presence of IPTG, and cells were then solubilized and put through several freeze-thaw cycles. Lysed cells were centrifuged at 10,000g × 5 min, and 20 µg of protein was electrophoresed on a 12% (w/v) polyacrylamide gel in the presence of SDS and stained with Coomassie blue (A). Presence of the DCC-His6 fusion protein in inclusion bodies was confirmed on a parallel gel of 20 µg of protein, by Western blot analysis using anti-His-C-terminal antibody with chemiluminescence detection (B). Arrows indicate the position (35.5 kDa) at which the DCC-His6 fusion protein elutes. Numbers on the right side of each panel are those from the molecular weight standards.

View larger version (94K): [in this window] [in a new window]   Fig. 2.   Demonstration of highly purified, bacterially expressed rat kidney mitochondrial DCC protein. The solubilized Ni2+-affinity column flow-through from clone 15 was analyzed by SDS-PAGE and stained with Coomassie blue. Electrophoresis of 10 µg of protein was carried out on a 10% (w/v) polyacrylamide gel in the presence of SDS. The right hand lane shows molecular weight standards.

Reconstitution of Recombinant Rat Kidney DCC-His₆ Protein. The partially purified DCC fraction was reconstituted into proteoliposomes to assess its transport activity with GSH and dicarboxylate substrates. Time courses for the uptake of GSH (0.05-10 mM; Fig. 3, A and B) and malonate (0.01-5 mM; Fig. 3, C and D) were determined in phosphate- and malate-containing proteoliposomes. In preloaded proteoliposomes, transport exhibited a characteristic spike, reflecting a heteroexchange of the trans- for cis-substrates. Thereafter, intravesicular radioactive substrate modestly decreased. Reconstituted DCC exhibited time- and concentration-dependent uptake of both GSH and malonate. For both substrates at nearly all concentrations tested, maximal intravesicular content of transported substrate was achieved by 2 min. Initial rates (after 2 min) and maximal intravesicular contents of malonate were approximately 5- to 6-fold higher than those for GSH.

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Time course of uptake of GSH (A and B) and malonate (C and D) in proteoliposomes reconstituted with bacterially expressed rat kidney mitochondrial DCC protein. Uptake of [3H]GSH or [14C]malonate was measured in proteoliposomes preloaded with 1 mM each of inorganic phosphate and malate. At indicated times, aliquots of incubation mixtures were mixed with 40 mM pyridoxal 5'-phosphate and centrifuged through Sephacryl S-100 mini-columns. Eluates were placed in scintillation vials for determination of radioactivity. Results are the means ± S.E. of measurements from four separate preparations.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Kinetics of GSH (A and B) and malonate (C and D) uptake in proteoliposomes reconstituted with bacterially expressed rat kidney mitochondrial DCC protein. Uptake of [3H]GSH or [14C]malonate was measured in proteoliposomes preloaded with 1 mM each of inorganic phosphate and malate. At indicated times (30 s to 4 min), aliquots of incubation mixtures were mixed with 40 mM pyridoxal 5'-phosphate and centrifuged through Sephacryl S-100 mini-columns. Eluates were placed in scintillation vials for determination of radioactivity. A/C. Initial rates (V) were calculated and are plotted versus substrate concentration (S). The curves for V versus S were generated by a second-order polynomial regression. Regression equations: A, y = 1.205x2 + 26.419x + 1.425 (r2 = 0.998); C, y = 33.223x2 + 264.973x + 44.422 (r2 = 0.981). B and D, Eadie-Hofstee plots (V versus V/S) for data in A and C, respectively. The lines were generated by linear regression. Regression equations: B, y = 8.386x + 263.420 (r2 = 0.912); D, y = 0.590x + 472.521 (r2 = 0.676). Results are the means ± S.E. of measurements from four separate preparations.

Mitochondrial GSH Transport Activity in Transfected NRK-52E Cells. To better explore the role of the DCC in GSH transport in an intact renal cell and to develop a strategy to increase mitochondrial GSH levels and protect cells from various forms of chemical-induced injury, NRK-52E cells were transiently transfected with the cDNA for the rat kidney mitochondrial DCC in pcDNA3.1/V5-His-TOPO. Mitochondrial fractions were prepared from three clones and were tested for the presence of the DCC-His₆ fusion protein by immunoblot analysis with antibody directed to the His₆ tag (Fig. 5). Whereas no reactivity was detected in the wild-type cells, a single, prominent band at approximately 35.5 kDa was detected in mitochondrial extracts from each of the three clones, indicating expression of the recombinant protein. The relative expression of the recombinant protein varied 1.72-fold, as judged by densitometry.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Expression of rat kidney mitochondrial DCC protein in NRK-52E cells. Mitochondria were isolated by differential centrifugation from wild-type (WT) and three clones of NRK-52E cells transiently transfected with the cDNA for rat kidney mitochondrial DCC-His6 recombinant protein. Equal amounts of protein (20 µg) were electrophoresed on SDS gels, transferred to nitrocellulose membranes, and reacted with the anti-His-C-terminal antibody. Detection was by chemiluminescence. A single protein band eluting on the SDS gel at 35.5 kDa was observed in each clone. Numbers above each lane are the relative density of the bands, normalized to the band with the lowest density, as determined with NIH Image version 1.6.2 software for Macintosh.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Accumulation of GSH and malonate in mitochondria of wild-type and transfected NRK-52E cells. Values for mitochondrial accumulation of 5 mM [3H]GSH (A) or 1 mM [14C]malonate (B) were determined after incubation for 2 min of suspensions of mitochondria isolated from wild-type NRK-52E cells or from three clones of NRK-52E cells transiently transfected with the cDNA for rat kidney mitochondrial DCC-His6 recombinant protein. Results are averages of duplicate measurements from single samples of wild-type cells (NRK-52E-WT) or transfectant clones (NRK-52E-rDCC+).

Influence of Overexpression of rDCC-His₆ on Sensitivity to Chemical-Induced Apoptosis. The influence of overexpression of the DCC protein on sensitivity of the NRK-52E cells to chemical-induced apoptosis was next examined. Both wild-type cells (NRK-52E-WT) and transfectants overexpressing rDCC-His₆ (NRK-52E-rDCC+) were incubated with 1 mM GSH for 24 h, followed by incubation for 1, 2, or 4 h without treatment, or with tBH (10 or 50 µM) or DCVC (50 or 200 µM), in the presence of 20 µM GSH. The 20 µM GSH was chosen to mimic the concentration of GSH in the extracellular or periplasmic space and is that to which renal cells are normally exposed (Lash and Jones, 1985).

View larger version (61K): [in this window] [in a new window]   Fig. 7.   Flow cytometry analysis of propidium iodide-stained wild-type NRK-52E cells treated with media, tBH, or DCVC. Confluent, wild-type NRK-52E cells (NRK-52E-WT) grown on 35-mm culture dishes were pretreated for 24 h with 1 mM GSH and then treated for up to 4 h with either media, 10 µM tBH, or 50 µM DCVC in the presence of 20 µM GSH. Cells were then harvested by trypsin/EDTA treatment, washed in sterile PBS, and fixed overnight in ethanol. Cells were then stained with propidium iodide and analyzed by flow cytometry with a FACSCalibur flow cytometer (BD Biosciences). Panels represent DNA histograms, with cell number (y-axis) plotted against DNA content or channel number (x-axis). Peaks from left to right represent apoptotic cells, cells in G0/G1, cells in S, and cells in G2/M. Values above the left peaks indicate the percentage of apoptotic cells. Insets, scatter profiles showing distribution of cells according to propidium iodide fluorescence intensity; y-axis, forward scatter; x-axis, side scatter. Cells outside the box are those that were excluded from the analysis due to aggregation.

View larger version (62K): [in this window] [in a new window]   Fig. 8.   Flow cytometry analysis of propidium iodide-stained NRK-52E cells transfected to overexpress rDCC-His6 and treated with media, tBH, or DCVC. Confluent, transfectant NRK-52E cells (NRK-52E-rDCC+) grown on 35-mm culture dishes were pretreated for 24 h with 1 mM GSH and then treated for up to 4 h with either media, 10 µM tBH, or 50 µM DCVC in the presence of 20 µM GSH. Samples were processed for FACS analysis as described in the legend to Fig. 7.

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Induction of apoptosis in wild-type and NRK-52E cells transfected to overexpress rDCC. Confluent, wild-type (NRK-52E-WT) and transfectant (NRK-52E-rDCC+) cells grown on 35-mm culture dishes were pretreated with 1 mM GSH and treated with media or toxicants in the presence of 20 µM GSH. Samples were processed for FACS analysis as described in the legend to Fig. 7. Values are from one representative series of samples and are the percentage of apoptotic cells plotted against incubation time.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Mitochondrial GSH status is a critical determinant of cellular energetics and function and plays a key role in determining susceptibility of cells to various forms of chemical or pathological injury. The ability to manipulate GSH status in this organelle can provide a greater understanding of precisely how mitochondrial GSH functions in various processes, including oxidative phosphorylation and apoptosis, but also a tool with which to modulate cellular injury upon exposure to various toxicants. The present communication describes one such approach in which one of the major mechanisms by which cells compartmentalize GSH is genetically altered to produce protection from two model toxicants.

Over the past several years, we have been studying the transport of GSH into renal proximal tubular mitochondria (McKernan et al., 1991; Chen and Lash, 1998; Chen et al., 2000). Transport of GSH from the cytoplasm into the mitochondrial matrix is the primary, if not sole, source of GSH for this subcellular compartment because of the absence of significant synthesis of GSH from its precursors within the mitochondrial matrix (Griffith and Meister, 1985; McKernan et al., 1991; Smith et al., 1996). Two electroneutral anion carriers, the DCC and OGC, were identified as being responsible for most, if not all, of the transport of GSH into renal cortical mitochondria (Chen and Lash, 1998; Chen et al., 2000). This conclusion was based on examination of substrate specificity and inhibitor studies. Although a partially purified preparation of mitochondrial anion carriers was reconstituted and used by others to study the transport of various dicarboxylates (Kaplan and Pedersen, 1985; Saint-Macary and Foucher, 1985; Bisaccia et al., 1988) and by us to study GSH transport (Chen et al., 2000), there are inherent problems or limitations with this experimental approach. First, the carrier proteins are present in the mitochondrial inner membrane at relatively low abundance, making it difficult to obtain sufficient amounts of material for experimentation. Second, of the various anion carriers of the inner membrane, the DCC protein, in particular, is relatively unstable in a purified form (Lançar-Benba et al., 1994), making it difficult to obtain sufficient amounts of pure protein suitable for functional assays or protein chemistry. The availability of the complete cDNA sequence from the rat (Fiermonte et al., 1998) affords us the opportunity to express the DCC protein in relatively large amounts, to study its catalytic function in greater detail and manipulate its molecular regulation to probe its physiological and toxicological function.

We showed previously that GSH transport into renal cortical mitochondria was insensitive to membrane potential (McKernan et al., 1991; Chen and Lash, 1998), which is consistent with the function of the electroneutral DCC and OGC. This suggests that GSH is transported by these carriers as a dianion. As discussed previously (Chen and Lash, 1998), the relatively alkaline pH of the mitochondrial matrix and the suggested presence of a microenvironment near the active site of the carrier proteins effectively lowers the pK_a of the thiol group so that a larger proportion of GSH molecules may be in the thiolate form.

In the present study, we expressed the full-length DCC cDNA from rat kidney mitochondria in bacteria as an N-terminal His₆ fusion protein to facilitate its isolation and detection with His₆-specific antibodies. DCC synthesis was induced with IPTG in a time-dependent manner and was extracted from inclusion bodies for fractionation on a Ni²⁺-Sepharose affinity column. Our results demonstrate that the partially purified DCC-His₆, reconstituted in lipid membranes, was functional. Function was shown by measurements of time and concentration dependence of GSH and malonate uptake. As we found in isolated mitochondria from rat kidneys (Chen and Lash, 1998) and in the enriched and reconstituted anion carrier preparation (Chen et al., 2000), transport of both substrates exhibits Michaelis-Menten kinetics and the rate of uptake and maximal accumulation of malonate are at least 5-fold greater than those for GSH. Nonetheless, the kinetics of GSH uptake calculated from the reconstituted, bacterially expressed DCC protein are reasonable compared with those observed previously in other in vitro model systems. The shape of the time courses for uptake of GSH and malonate (Fig. 3), which exhibited maxima at 2-min time points and slight decreases thereafter, is consistent with that which we and others typically observe in artificial proteoliposome vesicles in which the transport process under investigation involves an exchange of substrates on opposite sides of the membrane; the slight overshoot is likely due to trans-stimulation of uptake.

Sensitivity of GSH and malonate uptake to various competitive and noncompetitive inhibitors provided additional evidence that recombinant DCC-His₆ was functionally identical to the protein detected in partially purified preparations from rat or rabbit kidney. This includes inhibition by butylmalonate, pyridoxal 5'-phosphate, and mercurials. Furthermore, GSH inhibited malonate uptake. With respect to the specificity of GSH uptake, -glutamyl amino acids and ophthalmic acid were good inhibitors, consistent with the properties we described previously for GSH transport into renal cortical mitochondria (McKernan et al., 1991; Chen and Lash, 1998). The properties that apply to dicarboxylate transport are also comparable with those previously reported in the literature (Bisaccia et al., 1985, 1988; Saint-Macary and Foucher, 1985; Kaplan and Pedersen, 1985; Fiermonte et al., 1993, 1998; Lançar-Benba et al., 1994; Kaplan et al., 1995; Iacobazzi et al., 1996; Palmieri et al., 1996; Kakhniashvili et al., 1997).

An important goal for the present study was to establish transport function upon transfection of the His-tagged DCC construct into an intact renal cell line, NRK-52E cells, derived from normal rat kidney proximal tubules. Unlike some other commonly used renal epithelial cell lines, basic cellular energetics and redox status in NRK-52E cells have not been characterized extensively. In a separate study (Lash et al., 2002), we provided baseline data on mitochondrial function and GSH transport and metabolism. With the exception of very low activity of brush-border membrane enzymes, including -glutamyltransferase, GSH metabolism and plasma membrane transport in NRK-52E cells were comparable with what are commonly observed in freshly isolated or primary cultures or rat renal proximal tubular cells. Notably, the NRK-52E cells have a low content of mitochondria, as judged by use of a mitochondria-specific fluorescent dye and confocal microscopy, and low activities of several mitochondrial dehydrogenases, of oxidative phosphorylation, and of GSH and malonate transport across the inner membrane, compared with those in freshly isolated or primary cultures of rat renal proximal tubular cells. This finding is certainly not uncommon in an immortalized, epithelial cell line. This "deficiency" in the NRK-52E cells in comparison with the in vivo renal proximal tubule may actually facilitate our studies because extracellular degradation of GSH, which can cause experimental artifacts in transport studies, is minimal in these cells.

Immunoblot analysis using an antibody directed against the C-terminal His₆ tag of our expressed protein confirmed successful transfection of the NRK-52E cells. More importantly, mitochondria isolated from transfectants exhibited significantly increased rates of uptake and higher maximal accumulation of both GSH and malonate than in those isolated from wild-type cells. The amount of stimulation of substrate uptake seemed to approximately correlate with the level of expression of the recombinant protein in the transfected NRK-52E cells. These observations were carried further by demonstrating that transfectants exhibited much less apoptosis compared with wild-type cells when incubated with tBH or DCVC, which are both oxidants and mitochondrial toxicants (Lash and Anders, 1987; Hagen et al., 1988; Lash and Tokarz, 1990; McKernan et al., 1991; Lash et al., 2002). Hence, overexpression of the DCC from rat kidney in a stable cell line derived from rat proximal tubule provides marked protection from oxidant-induced apoptosis. This experimental approach allows us to manipulate the mitochondrial thiol/disulfide redox and GSH status, thereby providing a novel tool with which to probe mitochondrial function and modulate cellular susceptibility to oxidants and other mitochondrial toxicants.

In summary, although we have confirmed that the bacterially expressed and reconstituted DCC protein exhibits identical function for transport of GSH and dicarboxylates as determined previously, the novelty of the present results is that we demonstrate function of a cloned and bacterially expressed mitochondrial transporter with GSH, which provides the framework for studies on the molecular regulation of and more controlled manipulation of mitochondrial GSH status. Furthermore, we show that NRK-52E cells exhibited increased rates of mitochondrial uptake of GSH and dicarboxylates when these cells are transiently transfected to overexpress the DCC protein and that these cells are relatively resistant to chemical-induced apoptosis.