Introduction

Top Abstract Introduction Results Discussion References

The multidrug resistance-associated protein (MRP1)² and P-glycoprotein are members of the ATP-binding cassette transporter family or ABC transporters (Gottesman and Pastan, 1993; Loe et al., 1996c). MRP1 and P-glycoprotein function as ATP-dependent export pumps with broad and sometimes overlapping substrate selectivities and are associated with multidrug resistance (Germann, 1996; Loe et al., 1996b). Both are integral membrane glycoproteins, although MRP1 differs from P-glycoprotein in that it has an MDR-like (or P-glycoprotein-like) core plus an additional N-terminal domain and cytoplasmic linker (Bakos et al., 1996). MRP1 was first identified in tumor cell lines that were multidrug resistant but did not express P-glycoprotein (Cole et al., 1992). Subsequent expression of MRP1 in drug-sensitive cell lines showed that MRP1 confers resistance to a range of chemotherapeutic drugs (Cole et al., 1994; Grant et al., 1994; Zaman et al., 1994). MRP1 is expressed in a number of cell lines, including human (Seetharaman et al., 1998), bovine (Huai-Yun et al., 1998), porcine (Gutmann et al., 1999), and murine (Kusuhara et al., 1998; Homma et al., 1999) capillary brain endothelial cells, in choroid-plexus endothelial cells (Rao et al., 1999), and in murine astrocytes (Declèves et al., 2000). MRP1 is also expressed in nonmalignant tissues, including brain, lung, testis, and peripheral blood (Kruh et al., 1995; Flens et al., 1996).

The transport mechanisms of MRP1 and P-glycoprotein differ; MRP1 transports multivalent organic anions with a preference for glutathione, sulfate, and glucuronide conjugates (Leier et al., 1994a; Müller et al., 1994; Jedlitschky et al., 1996; Loe et al., 1996a,b). Also glutathione disulfide and some unconjugated compounds are MRP1 substrates, although the latter require the presence of glutathione for transport (Rappa et al., 1997; Loe et al., 1998; Renes et al., 1999). In contrast, P-glycoprotein preferentially transports unconjugated drugs, which vary both structurally and functionally, including the chemotherapeutic vinca alkaloids, etoposide, and paclitaxel. These agents are generally neutral or basic (Shinkel, 1997).

Monochlorobimane [syn-(ClCH₂,CH₃)-1,5-diazabicyclo-[3.3.0]-octa-3,6-dione-2,8-dione; mClB] is a nonfluorescent compound that readily crosses the cell membrane. Once inside the cell, mClB is conjugated with glutathione in a reaction catalyzed by glutathione transferases to the highly fluorescent conjugate B-SG (Rice et al., 1986; Shrieve et al., 1988). Preliminary studies with primary cultures of rat striatal and mouse cortical neurons revealed that B-SG is formed within these cells and that the fluorescent product appeared to be effluxed or transported out of these cells over time. The objective of this study was to further characterize the efflux of B-SG in primary cultures of rat striatal neurons and mouse cortical neurons. The data presented demonstrate the presence of a transporter functionally similar to MRP1. To our knowledge, this is the first report of an MRP1-like transporter in neurons.

Experimental Procedures

Materials. MK-571 was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). Monochlorobimane and monobromobimane were obtained from Molecular Probes (Eugene, OR). Verapamil, 2,4-dinitrofluorobenzene, ethacrynic acid, probenecid, glutathione, 3-O-methyl-D-glucopyranose, D-glucose, cytosine-arabinose, sodium acetate trihydrate, bovine serum albumin, HEPES, polyethyleneimine, and laminin were obtained from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum was obtained from Hyclone (Logan, UT). Neurobasal medium, L-glutamine, L-glutamic acid, B27 serum-free supplement, N₂ supplement, fetal bovine serum, trypsin/EDTA, Fungizone, gentamicin, Hanks' balanced salt solution (calcium and magnesium free), and Dulbecco's modified Eagle's medium were purchased from Invitrogen (Carlsbad, CA). Glass coverslips and 6-well, 35-mm culture dishes (Corning) were purchased from VWR Scientific Products (Rochester, NY). Goat polyclonal antibodies raised against epitopes that map 1) near the carboxy terminus of human MRP1, 2) the amino terminus of human MRP1, and 3) the carboxy terminus of mouse MRP1 and rabbit polyclonal antibodies raised against an epitope that maps near the carboxy terminus of Mdr-1 of human origin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Donkey anti-goat IgG linked to horseradish peroxidase and goat anti-rabbit IgG linked to horseradish peroxidase were purchased from Bio-Rad (Hercules, CA). All other chemicals were obtained from commercial sources and were of the highest purity available.

Primary Cultures of Rat Striatal Neurons and Mouse Cortical Neurons. All procedures for animal use were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rat striatal neurons were obtained from the neurostriatal region of embryonic day-17 rat pups (Greene et al., 1998). Freshly isolated neurons were plated either on laminin-coated, 25-mm glass coverslips at a density of 4 × 10⁵ cells/coverslip for image analysis or on laminin-coated, 6-well culture dishes (35-mm-diameter well) at a density of 1 × 10⁶ cells/well for biochemical analysis. All cultures were incubated at 37°C in a humidified atmosphere of 95% O₂, 5% CO₂. Primary striatal cultures were grown in serum-containing feeding medium (10% fetal bovine serum, 35 mM glucose, 2 mM L-glutamine, 2.5 µg/ml Fungizone, and 50 µg/ml gentamicin in Dulbecco's modified Eagle's medium; final osmolality, ~310 mOsm/l) for up to 5 days in culture; the cultures were then treated with cytosine arabinoside (5 µM) for 2 days and were incubated in serum-free feeding medium (35 mM glucose, 0.5 mM L-glutamine, 27.5 mM NaCl, 2.5 µg/ml Fungizone, 50 µg/ml gentamicin, and N₂ supplement in Neurobasal medium; final osmolality, ~310 mOsm/l) for up to 16 days in culture. Glial contamination was routinely less than 10%, as determined by counting individual cells.

Formation of B-SG in Primary Cultures. Mouse cortical neurons were prepared as described above on coverslips, which were mounted in a modified Skyes-Moore chamber (Bellco, Vineland, NJ) and incubated in loading buffer (50 µM mClB, 10 µM glutathione, 140 mM NaCl, 10 mM glucose, 5 mM KCl, 1.8 mM CaCl₂, 5 mM HEPES, and 1 g/l bovine serum albumin, pH 7.4). After 15 min, the coverslips were washed with efflux buffer (loading buffer lacking mClB), and images were acquired with a Nikon Diaphot inverted microscope (40×, oil-immersion objective) fitted with a Nikon N50 camera. A computer-controlled scanning monochromator (Delta Scan 1; Photon Technology Inc., Princeton, NJ) was used to select the excitation (385 nm) and emission (485 nm) wavelengths. When indicated, neurons were incubated with ethacrynic acid (50 µM in efflux buffer) for 10 min before loading with mClB.

Efflux Experiments. Rat striatal neurons and mouse cortical neurons were grown in 6-well culture plates, as indicated above. The culture medium was removed, and neurons were incubated with 2 ml of loading buffer at 37°C in a Dubnoff metabolic shaker (VWR Scientific Products) at setting 2. After 15 min, the loading buffer was removed and replaced with 2 ml of efflux buffer, and the culture plates were returned to the metabolic shaker. At the indicated times 1 ml of efflux buffer was removed from the wells for determination of B-SG concentrations in the medium, and an additional 200 µl of the efflux buffer was removed for determination of lactate dehydrogenase activities (Decker and Lohmann-Matthes, 1988), except that the reactions were done in 1-cm path length cuvettes. The remaining efflux buffer was removed by aspiration, and the neurons were lysed by addition of 1 ml of distilled water followed by sonication for 60 s in a Branson 2200 sonicating bath (Branson Ultrasonic Corp., Danbury, CT). Ten microliters of perchloric acid (69-72%) was added to each of the wells, and the resulting mixture was transferred to 1.7-ml plastic centrifuge tubes. The efflux buffer (1 ml) was also treated with 10 µl of perchloric acid, and precipitated proteins in the neuronal lysate and efflux buffer were removed by centrifugation. The amounts of B-SG in the resulting supernatants were determined by measuring fluorescence intensity (excitation wavelength, 385 nm; emission wavelength, 485 nm) with a PerkinElmer LS-5 fluorescent spectrophotometer (Norwalk, CT).

Western Blot Experiments. Primary cultures of rat striatal and mouse cortical neurons in 6-well format were washed twice with ice-cold PBS (0.15 M NaCl and 10 mM sodium phosphate, pH 7.5). Cells were lysed with 150 µl of SDS buffer (10% SDS, 2.5 M sucrose, 0.2 M Tris, and 0.02 M EDTA, pH 6.8); the lysates were transferred to centrifuge tubes and briefly sonicated. Protein concentrations were determined by the method of Lowry et al. (1951). -Mercaptoethanol and bromophenol blue were added to the cell lysates to a final concentration of 3 mM and 0.02%, respectively. Protein samples (25-120 µg) were heated in a boiling water bath for 5 min, analyzed by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Bio-Rad) (Laemmli, 1970). The presence of MRP1 or P-glycoprotein was detected with a modification of the method provided by the antibody manufacturer. Briefly, the nitrocellulose membranes were blocked with 3% dry milk in PBS for 1 h with gentle shaking at room temperature. The membranes were then incubated with the manufacturer's recommended dilution of primary antibody in 3% dry milk in PBS overnight. The next day, the membranes were rinsed with three changes of PBS prior to incubation for 45 min at room temperature with the appropriate horseradish peroxidase-linked second antibody also diluted in 3% dry milk in PBS (i.e., donkey anti-goat IgG conjugate was used for the MRP1 antibodies, and goat anti-rabbit IgG-horseradish peroxidase conjugate was used for the P-glycoprotein antibody). After incubation with secondary antibody, the membranes were rinsed with five changes of PBS, and immunolabeled proteins were visualized with an ECL chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's directions. The positive control for MRP1 included rat lung or kidney homogenates; the positive control for P-glycoprotein was a ZR-75-1 whole-cell extract (human breast carcinoma cell line; Santa Cruz Biotechnology).

Data Analysis. Data were analyzed with the two-tailed Student's t test or analysis of variance. A probability of p < 0.05 was selected for acceptance or rejection of the null hypothesis. Statistical analyses were done with GraphPad Prism, version 3.00 (GraphPad Software, Inc., San Diego, CA). Standard curves of B-SG were prepared by fitting data to a weighted (1/Y) linear regression equation.

	Results

Top Abstract Introduction Results Discussion References

Time-Dependent Efflux of B-SG. To visualize the formation of B-SG, mouse cortical neurons and rat striatal neurons plated on coverslips were loaded with mClB, and fluorescent and phase-contrast images were acquired, as described under Experimental Procedures. Rat striatal neurons and mouse cortical neurons loaded with mClB were highly fluorescent, indicating the intracellular formation of the B-SG conjugate (Figs. 1B and 2B). In contrast, rat striatal and cortical neurons incubated with ethacrynic acid prior to loading with mClB failed to show detectable fluorescence (Figs. 1D and 2D).

View larger version (61K): [in this window] [in a new window]   Fig. 1.   Formation of B-SG in primary cultures of mouse cortical neurons. Neurons were isolated and cultured on glass coverslips, as described under Experimental Procedures. The neurons were loaded with 50 µM mClB for 15 min prior to imaging. A, phase-contrast image of mClB-loaded cells. B, fluorescence image acquired from the same field. C, phase-contrast image of mClB-loaded cells incubated with ethacrynic acid. D, fluorescent image from the same field. Scale bar, 10 µm. Arrows show the same neuron in A and B or in C and D.

View larger version (66K): [in this window] [in a new window]   Fig. 2.   Formation of B-SG in primary cultures of rat striatal neurons. Neurons were isolated and cultured on glass coverslips, as described under Experimental Procedures. The neurons were loaded with 50 µM mClB for 15 min prior to imaging. A, phase-contrast image of mClB-loaded cells. B, fluorescence image acquired from the same field. C, phase-contrast image of mClB-loaded cells incubated with ethacrynic acid. D, fluorescent image from the same field. Scale bar, 10 µm. Arrows show the same regions in A and B or in C and D.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Efflux of B-SG in rat striatal neurons (A) and mouse cortical neurons (B). Cultures of rat striatal neurons and mouse cortical neurons were incubated with 50 µM mClB for 15 min. At time 0, the medium was removed and replaced with medium that lacked mClB, and the amount of B-SG excreted into the medium (triangles) and that remaining in the neurons (squares) was measured. Total amounts of B-SG (B-SG in the medium plus B-SG in neurons) are also shown (circles). Data represent means ± S.E.M. for three separate wells.

Effect of Inhibitors on B-SG Efflux. The efflux of B-SG from rat striatal neurons was measured in the presence and absence of potential inhibitors. Neurons were loaded with mClB (50 µM) in the presence or absence of probenecid (100 µM), verapamil (10 µM), or MK-571 (10 µM); control cultures were incubated with vehicle only. After 15 min, the loading buffer was removed, and the cultures were incubated for another 40 min with the inhibitors. MK-571 caused a significant decrease in the efflux of B-SG in both rat striatal and mouse cortical neurons compared with controls, whereas probenecid caused a significant decrease in the efflux of B-SG from rat striatal neurons, but not from mouse cortical neurons compared with controls (Fig. 4). Verapamil failed to inhibit the efflux of B-SG in both rat striatal and mouse cortical neurons (Fig. 4). There was no difference in total amount of B-SG formed between control cells and cells incubated with verapamil or MK-571 in rat striatal neurons, although there was a small, but significant (p < 0.05), decrease in the total amount of B-SG formed in rat striatal neurons incubated with probenecid. In mouse cortical neurons, there was no difference in total amounts of B-SG formed between control cells and cells incubated with probenecid and verapamil. A small, but significant (p < 0.05), decrease in the total amounts of B-SG formed was observed in cells incubated with MK-571. There was no release of lactate dehydrogenase activity in rat striatal or mouse cortical neuron cultures incubated with inhibitors (data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effect of inhibitors on the efflux of B-SG from rat striatal neurons (A) and mouse cortical neurons (B). Cultures of rat striatal neurons and mouse cortical neurons were incubated with 50 µM mClB for 15 min in the presence or absence of probenecid (100 µM), verapamil (10 µM), or MK-571 (10 µM). Control cultures were incubated with vehicle. At time 0, the medium was removed and replaced with medium that lacked mClB but contained inhibitors, as indicated. Cultures were incubated at 37°C, and the amount of B-SG transported into the medium was measured after 40 min. Data are shown as means ± S.E.M. for three separate wells. *p < 0.05 compared with control.

Effect of Temperature and ATP Concentration on B-SG Efflux. Neurons were loaded with mClB (50 µM) in loading buffer. After 15 min, the loading buffer was removed and the neurons were incubated with either efflux buffer containing glucose or efflux buffer containing 3-O-methyl-glucopyranose, sodium fluoride, and sodium azide; and the neurons were further incubated at either 37 or 20°C for 40 min. There was a significant decrease in the efflux of B-SG from both rat striatal and mouse cortical neurons incubated at 20°C compared with cells incubated at 37°C (Fig. 5). In addition, there was a significant decrease in efflux of B-SG when neurons were incubated in the presence of reduced ATP concentrations compared with constitutive ATP concentrations (Fig. 5). Total amounts of B-SG formed were lower (13 ± 2.3%, p < 0.05, and 22 ± 5.9%, p < 0.05) in rat striatal neurons and mouse cortical neurons, respectively, incubated in the presence of reduced ATP concentrations compared with those incubated in the presence of constitutive ATP concentrations. In addition, total amounts of B-SG were 20 ± 2.9% lower in rat striatal neurons incubated at 20°C compared with those incubated at 37°C; there was no difference in total amounts of B-SG in mouse cortical neurons incubated at 20°C compared with those incubated at 37°C. Finally, there was no release of lactate dehydrogenase activity in cells incubated in the presence of reduced ATP concentrations or at 20°C (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of reduced ATP concentrations and temperature on B-SG efflux from rat striatal neurons (A) and mouse cortical neurons (B). Cultures of rat striatal neurons and mouse cortical neurons were incubated with 50 µM mClB for 15 min. At time 0, the medium was removed and replaced with efflux buffer that contained glucose (10 mM) (+ATP) or efflux buffer that contained 10 mM 3-O-methyl-D-glucopyranose, 10 mM NaF, and 10 mM NaN3 (ATP). Cultures were then incubated at 37 or 20°C, and the amount of B-SG transported into the medium was measured after 40 min. Data are shown as means ± S.E.M. for three separate wells. Analysis of variance: effect of ATP concentration and temperature in both rat striatal and mouse cortical neurons, p < 0.0001.

Western Blot Analysis. Protein lysates of primary cultures of rat striatal neurons and mouse cortical neurons were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed separately with anti-MRP1 and anti-P-glycoprotein antibodies. No 190-kDa proteins were detected in either neuronal lysate (data not shown), even when loaded with as much as 120 µg of protein per lane. The positive controls for MRP1, i.e., rat lung and kidney homogenates, showed a protein at the appropriate molecular mass (data not shown). In contrast to the anti-MRP1 antibodies tested, the anti-P-glycoprotein antibody showed the presence of a 170-kDa protein in rat striatal neuron lysates and in the positive control (ZR-75-1 extract), but not in the mouse cortical neuron lysates. A 220-kDa protein was detected in the mouse cortical neuron lysate and may represent a glycosylated form of mouse P-glycoprotein.

	Discussion

Top Abstract Introduction Results Discussion References

In the current study, the pool of glutathione in rat striatal neurons and mouse cortical neurons was labeled with mClB, which undergoes a glutathione transferase-catalyzed reaction to form the stable, fluorescent conjugate B-SG (Rice et al., 1986; Shrieve et al., 1988). The intensity of the resulting fluorescence is dependent on the initial glutathione concentration as well as on the glutathione transferase isozymes present in the cell. The µ-class neutral transferase catalyzes the reaction of mClB with glutathione in rat tissues (Cook et al., 1991). Preliminary fluorescence microscopy studies of single neurons showed that B-SG was rapidly formed in these cells and that conjugate formation was inhibited by incubation of neurons with ethacrynic acid (Figs. 1 and 2), which depletes cytosolic and mitochondrial glutathione concentrations (Meredith and Reed, 1982; Reed, 1990). These results are in agreement with those of Tauskela et al. (2000), who showed that fluorescence staining with mClB in rat cortical neurons is inhibited with diethyl maleate or DL-buthionine-(S,R)-sulfoximine. The preliminary studies also showed, however, that the fluorescence attributable to B-SG decreased in a time-dependent manner (data not shown). The loss of intracellular fluorescence was not due to photobleaching of the glutathione conjugate as evidenced by the stability of the authentic standard when continuously exposed to its excitation wavelength (data not shown).

The objective of this study was to characterize the mechanism of the loss of B-SG from rat striatal and mouse cortical neurons and to identify transporters or metabolic pathways that might be involved. Studies with primary cultures of rat striatal and mouse cortical neurons showed that mClB entered the neurons and was rapidly conjugated with glutathione during the 15-min loading period. There was little variability in the levels of intracellular B-SG immediately after the loading period, suggesting uniform loading efficiency from well to well (Fig. 3) and preparation to preparation (data not shown). The size of the glutathione pool in rat striatal neurons found in the present studies was about 2-fold higher than that reported by others (Dringen et al., 1999; Wang and Cynader, 2000). This difference may be attributed to the use of rat neurons after different days in culture or to the use of different culture conditions. Dringen et al. (1999) demonstrated that neuronal glutathione concentrations could be increased by culturing neurons in the presence of glutathione precursors or by transiently coculturing the neurons with astrocytes. The neuronal culture medium used in the present studies contained cysteine, a glutathione precursor. In addition, in the current study, total amounts of B-SG continued to increase after removal of the mClB-containing loading buffer in both rat striatal and mouse cortical neuron cultures (Fig. 3). Hence, it is likely that the intracellular glutathione is not totally conjugated during the 15-min incubation period and that conjugation with intracellular mClB continues after removal of the loading buffer. This possibility is supported by the results of Homma et al. (1999), who also observed continued B-SG formation in a mouse brain capillary endothelial cell line (MBEC4) after removal of mClB.

mClB may react with thiols other than glutathione, such as cysteine or protein thiols, to form a fluorescent product. However, HPLC analysis of the neuronal lysates and medium at increasing times after the removal of mClB showed a single fluorescent product that cochromatographed with authentic B-SG. Although these data clearly indicate that mClB reacted only with glutathione, they also exclude the possibility of secondary metabolism of B-SG to the cysteine or mercapturic acid conjugate with subsequent efflux into the medium. Hence, B-SG does not undergo further metabolism and is transported intact out of the neurons into the medium: 50 and 40% of the B-SG was transported out of rat striatal neurons and mouse cortical neurons, respectively, within 30 min.

Of the inhibitors tested, MK-571, a leukotriene D₄ receptor antagonist and an MRP1-selective inhibitor (Gekeler et al., 1995), inhibited the efflux of B-SG in both rat striatal and mouse cortical neurons, indicating that a MRP1-like transporter was involved in the efflux of B-SG. Although MK-571 was the most effective inhibitor, probenecid, an organic anion transport inhibitor, also inhibited the efflux of B-SG from rat striatal neurons. This observation agrees with that of Leier et al. (1994b), who showed inhibition of leukotriene C₄ transport into membrane vesicles prepared from mastocytoma cells by both probenecid and MK-571. In the present study, probenecid did not inhibit the efflux of B-SG from mouse cortical neurons, possibly due to different transport kinetics in mouse cortical neurons. Verapamil, an inhibitor of P-glycoprotein-dependent transport, had no effect on the efflux of B-SG in either neuronal culture system. Total amounts of B-SG formed were slightly reduced in rat striatal neurons incubated with probenecid and in mouse cortical neurons incubated with MK-571. The reasons for this decrease are unclear, but probenecid and MK-571 may interfere with either the uptake of mClB into neurons or conjugation of mClB with glutathione; this hypothesis does not, however, explain the observed differences between rat striatal and mouse cortical neurons.

There was a significant decrease in the efflux of B-SG in both rat striatal and mouse cortical neurons when neurons were incubated at 20°C compared with 37°C or in the presence of reduced ATP concentrations. These results indicate an active, ATP-dependent efflux process and further support the role of an ABC transporter, such as MRP1, in the efflux of B-SG. In these studies, total amounts of B-SG were lower in striatal and cortical neurons incubated at lower temperatures compared with control, which may be explained by lowered glutathione transferase activities at lower temperatures.

Whereas the present study showed that primary cultures of cortical and striatal neurons possess a transporter functionally similar to MRP1, immunoreactive MRP1 protein could not be detected by Western blot analysis in mouse cortical and rat striatal neuronal lysates. Two of the polyclonal antibodies tested in the current study were raised against peptides that map near the amino terminus of human MRP1 and the carboxy terminus of human MRP1, and the third was raised against a peptide that maps near the carboxy terminus of the mouse MRP1. Hence, the form of MRP expressed in the neurons of mice and rats must differ in structure from MRP1. At least three distinct MRP proteins have been identified in the rat and at least seven distinct MRP proteins have been identified in humans, some of which have less than 40% homology (Borst et al., 2000). Furthermore, Gutmann et al. (1999) and Seetharaman et al. (1998) showed that expression of MRP1 protein in brain capillary endothelial cells increases with increasing days in culture. In the current study, mouse cortical neurons and rat striatal neurons were used after 5 to 7 and 14 to 16 days in culture, respectively. Thus, rat striatal and mouse cortical neurons may express detectable levels of MRP1 after longer days in culture, although this was not investigated. In contrast to MRP1, P-glycoprotein expression was detected in primary cultures of rat striatal neurons and possibly in mouse cortical neurons. Verapamil failed to affect the efflux of B-SG in either culture system, indicating that although P-glycoprotein is present in primary cultures of rat striatal and mouse cortical neurons, it does not play a significant role in the efflux of B-SG.

In conclusion, data presented herein demonstrate the presence of an ABC-like transporter for B-SG and probably other glutathione conjugates in primary cultures of mouse cortical neurons and rat striatal neurons that has functional features consistent with those of MRP1. To our knowledge, this is the first report of MRP1-like function in nonmalignant cells of the central nervous system other than astrocytes or the epithelial cells lining the blood-brain barrier. Finally, we propose that this MRP1-like transporter may act as a second line of defense after the blood-brain barrier to limit the exposure of neurons to toxic compounds. MRP1 expression in neurons may, however, also limit the effectiveness of agents used in the chemotherapy of neuroblastomas, some of which contain high MRP1 expression (Norris et al., 1996).