The Journal of Neuroscience, December 15, 1999, 19(24):10767–10777 Compromised Glutamate Transport in Human Glioma Cells: Reduction–Mislocalization of Sodium-Dependent Glutamate Transporters and Enhanced Activity of Cystine–Glutamate Exchange Zu-Cheng Ye,1 Jeffrey D. Rothstein,2 and Harald Sontheimer1 1Department of Neurobiology, The University of Alabama at Birmingham, Birmingham, Alabama 35294, and 2Department of Neurology, Johns Hopkins University, Baltimore, Maryland 21287 1 Elevated levels of extracellular glutamate ([Glu]o ) can induce 50% of glutamate transport was Na -independent and medi- 2 seizures and cause excitotoxic neuronal cell death. This is ated by a cystine–glutamate exchanger (system xc ). Extracel- normally prevented by astrocytic glutamate uptake. Neoplastic lular L-cystine dose-dependently induced glutamate release transformation of human astrocytes causes malignant gliomas, from glioma cells. Glutamate release was enhanced by extra- which are often associated with seizures and neuronal necrosis. cellular glutamine and inhibited by (S)-4-carboxyphenylglycine, Here, we show that Na 1-dependent glutamate uptake in gli- which blocked cystine–glutamate exchange. These data sug- oma cell lines derived from human tumors (STTG-1, D-54MG, gest that the unusual release of glutamate from glioma cells is D-65MG, U-373MG, U-251MG, U-138MG, and CH-235MG) is caused by reduction–mislocalization of Na 1-dependent gluta- up to 100-fold lower than in astrocytes. Immunohistochemistry mate transporters in conjunction with upregulation of cystine– and subcellular fractionation show very low expression levels of glutamate exchange. The resulting glutamate release from gli- the astrocytic glutamate transporter GLT-1 but normal expres- oma cells may contribute to tumor-associated necrosis and sion levels of another glial glutamate transporter, GLAST. How- possibly to seizures in peritumoral brain tissue. ever, in glioma cells, essentially all GLAST protein was found in cell nuclei rather than the plasma membrane. Similarly, brain Key words: brain tumor; glutamate transporter; glutamate 2 tissues from glioblastoma patients also display reduction of release; cystine–glutamate exchange; system xc ; excitotoxic- GLT-1 and mislocalization of GLAST. In glioma cell lines, over ity; epilepsy Glutamate is the primary excitatory amino acid neurotransmitter homeostasis (Bergles and Jahr, 1998; Magistretti et al., 1999). in the mammalian CNS. Maintenance of low extracellular gluta- Astrocytes can convert transported glutamate to glutamine, mate concentrations ([Glu]o) is critical to ensure synaptic trans- which is then released as precursor for neuronal synthesis of mission and to prevent neurotoxicity (Choi, 1988; Nicholls and neurotransmitter glutamate (Rothstein and Tabakoff, 1984; Attwell, 1990). Toxicity from elevated [Glu]o (excitotoxicity) has Waniewski and Martin, 1986; Laake et al., 1995; Sibson et al., been suggested to be involved in a wide spectrum of acute and 1997). chronic nervous system diseases (Olney, 1982; Choi, 1988; Lipton Under disease conditions, glial glutamate transport can be and Rosenberg, 1994). In the healthy brain, abnormal rises of impaired and may contribute to the elevation of [Glu]o. For [Glu]o to excitotoxic levels are prevented by the activities of 1 instance, GLT-1 expression is severely decreased in the motor Na -dependent glutamate transporters. To date, five transporter cortex and spinal cord of patients with the sporadic form of subtypes have been cloned (Kanai and Hediger, 1992; Pines et al., amyotrophic lateral sclerosis, leading to elevations of excitatory 1992; Storck et al., 1992; Fairman et al., 1995; Arriza et al., 1997). amino acids in the CSF (Rothstein et al., 1990, 1995). Direct links These include the glutamate transporters GLAST and GLT-1, between compromised glutamate transport and neurotoxicity which are primarily expressed by astrocytes (Rothstein et al., have been demonstrated through knock-out experiments. Sup- 1994; Torp et al., 1994; Lehre et al., 1995) and appear to be the pression of the astrocytic glutamate transporters GLT-1 and most abundant glutamate transporters in brain (Lehre and Dan- GLAST by antisense oligonucleotides caused drastic rises in bolt, 1998). Because astrocytes are in close proximity to synapses, [Glu]o, sufficient to induce neuronal damage (Rothstein et al., they are believed to play a pivotal role in maintaining glutamate 1996). GLT-1 knock-out mice undergo lethal spontaneous epilep- tic seizures and display increased susceptibility to acute brain Received May 26, 1999; revised Sept. 7, 1999; accepted Oct. 6, 1999. injury (Tanaka et al., 1997), whereas mice in which the neuronal This research was supported by National Institutes of Health Grants R01-NS- 31234 and R01-NS-36692 and American Cancer Society Grant RPG-97-083. We glutamate transporter EAAC-1 had been knocked out developed thank the Brain Tumor Tissue Bank (London, Ontario, Canada), the National neither neurodegeneration nor epilepsy (Peghini et al., 1997). Cancer Institute of Canada, and the Brain Tumor Foundation of Canada for providing tumor samples. We are grateful to Dr. Susan Lyons and Jeffrey O’Neal for Elevation of [Glu]o may not only arise from reduced expression preparing tissue sections. of glutamate transporter but can also be caused by the reversed Correspondence should be addressed to Dr. Harald Sontheimer, Department of operation of glutamate transport or by other pathways that can Neurobiology, The University of Alabama at Birmingham, 1719 6th Avenue South, 1 CIRC 545, Birmingham, AL 35294. E-mail: [email protected]. mediate glutamate efflux. All the cloned Na -dependent trans- 1 1 Copyright © 1999 Society for Neuroscience 0270-6474/99/1910767-11$05.00/0 porters are driven by the electrochemical gradients for Na ,K , 10768 J. Neurosci., December 15, 1999, 19(24):10767–10777 Ye et al. • Glutamate Uptake and Release in Glioma Cells 1 and H to transport glutamate against its steep transmembrane changed twice a week, and astrocytes were used after 10 d in culture, at . gradient (Attwell et al., 1993; Zerangue and Kavanaugh, 1996a). which time 90% of cells were GFAP-positive and essentially free of neurons. Compromising the ionic environment can lead to reversal of Glutamate–aspartate uptake. Uptake procedures were similar to those transport. Then the millimolar cytoplasmic concentrations of we have described previously (Ye and Sontheimer, 1996) with minor 3 glutamate in astrocytes (Hertz et al., 1988; Levi and Patrizio, modifications. H-D-aspartate and D-aspartate as stable glutamate ana- 1 1992) can become a significant source for nonvesicular glutamate logs were used to study high-affinity, Na -dependent glutamate uptake. 3 release, which may contribute to neuronal injury (Szatkowski et In some cultures, results were compared with H-glutamate uptake. The solution for uptake consisted of (in mM): 125 NaCl, 3.0 KCl, 2.0 CaCl , al., 1990; Longuemare and Swanson, 1995). In addition to rever- 2 1.25 NaH2PO4, 23 NaHCO3, 10 glucose, and 2.0 MgSO4, warmed to 37°C sal of transport, swelling-activated anion channels have also been and saturated with 5% CO2–95% O2. For experiments dealing with 1 shown to mediate efflux of amino acids, including glutamate Na -independent glutamate–aspartate uptake, NaCl was replaced by 1 (Kimelberg and Mongin, 1998). Furthermore, an Na - choline chloride or N-methyl-D-glucamine, NaH2PO4 was replaced by 2 independent cystine–glutamate exchange (equal to system x in KH2PO4 with KCl lowered by 1.25 mM, and NaHCO3 was replaced with c triethylammonium bicarbonate (Kimelberg et al., 1989). Cells were fibroblast) (Bannai and Kitamura, 1980), which has recently been washed twice with the above uptake solution before experiments com- cloned (Sato et al., 1999), is expressed by a variety of cell types menced. The above uptake solution supplemented with 0.1 mM 3 (Watanabe and Bannai, 1987; Cho and Bannai, 1990; Murphy et D-aspartate and 0.5 mCi/ml H-D-aspartate was then added for 10 min. al., 1990; Piani and Fontana, 1994). Intracellular glutamate con- Uptake was terminated by three washes with ice-cold PBS. Cells were then dissolved in 0.3 N NaOH and aliquoted. 3H activity was detected in centrations in astrocytes are at levels above 1 mM (Hertz et al., a liquid scintillation counter (Beckman Instruments, Fullerton, CA) and 1988), whereas L-cystine levels are presumably much lower be- normalized to protein contents as determined by the Bio-Rad protein cause intracellular L-cystine is readily reduced to L-cysteine (Ban- assay kit (Bio-Rad, Hercules, CA). Background radioactivity was deter- nai and Kitamura, 1980). Consequently, the transmembrane glu- mined in the same manner as uptake but with the presence of 10 mM tamate gradient likely favors the efflux of glutamate in exchange unlabeled glutamate or D-aspartate and was subtracted from the uptake reading. for cystine. L-Cystine is required for the synthesis of glutathione 3 3 To determine the kinetics of uptake, H-glutamate and H-D-aspartate (Sato et al., 1998). uptake was performed in the presence of 5.0–400 mM glutamate or Unlike most neurons, glial cells can proliferate in response to D-aspartate, respectively. Apparent Vmax and Km were determined from injury or under neoplastic conditions. The vast majorities of the double reciprocal plot of uptake rate versus substrate concentration (Lineweaver–Burk plot) or from Eadie–Hofstee plots. primary brain neoplasms derived from glial cells and are collec- 35 L-cystine uptake. S-L-cystine uptake was performed in a way similar tively called gliomas. These tumors are rapidly expanding and are to glutamate uptake. Because intracellular cystine can be quickly re- often associated with seizures (Paillas, 1994). We show here that duced to cysteine and released back into the media (Bannai and Ishii, 1 glioma cells show much reduced cell surface expression of Na - 1982), the time course of uptake was shortened toa3minperiodto 35 dependent glutamate transporters thereby compromising their minimize loss of intracellular S. For kinetic measurements, L-cystine m ability to maintain glutamate homeostasis. The resulting gluta- concentrations in the uptake media ranged from 15 to 400 M.