Journal of Neurochemistty Raven Press, Ltd., New York 0 1990 International Society for Neurochemistry
Uptake of Glutamate and Cystine in C-6 Glioma Cells and in Cultured Astrocytes Yukie Cho and Shiro Bannai Department of Biochemistry, Tsukuba University Medical School, Tsukuba, Ibaraki, Japan
Abstract: The uptake of glutamate in rat glioma C-6 cells and cultured astrocytes derived from rat cerebral hemispheres was found to be mediated by a Na+-dependent and a Na+independent system. The Na+-dependent system was inhibited by aspartate and was consistent with the commonly ocThe Na+-independent curring system designated system X ~ .G system was inhibited by cystine and was consistent with system G- described in various types of cells in the periphery. It was also found that quisqualate selectively and competitively interfered with the Na'hdependent glutamate uptake. In C-6 cells, the glutamate uptake via systems XiG and x; accounted for approximately 35% and 55% of the total uptake, respectively, at 0.05 mM glutamate. In cultured astrocytes,
the glutamate uptake via system XiGwas very potent, whereas the uptake via system x i was relatively weak and its contribution to the total uptake of glutamate seemed almost negligible. However, in both C-6 cells and astrocytes, system was necessary for the uptake of cystine, another substrate of system x i . Cystine in the culture medium was an essential precursor of glutathione, and the inhibition of the cystine uptake by excess glutamate as a competitor led to a severe deficiency in glutathione, followed by cell degeneration. Key Words: Glutamate transport-Cystine-Glutathione-C-6 glioma cells-Astrocytes. Cho Y. and Bannai S. Uptake of glutamate and cystine in C-6 glioma cells and in cultured astrocytes. J. Neurochem. 55, 209 1-2097 (1 990).
Glutamate is a primary candidate for a role as an excitatory transmitter, and a glutamine cycle has been proposed as a mechanism for maintaining neuronal glutamate levels in the face of continual loss by transmitter release (Van den Berg and Garfinkel, 1971). According to this cycle, glutamate released from neurons as a transmitter is accumulated by glia and converted to glutamine. The glutamine is then returned to the neurons, where it provides renewed substrate for neurotransmitter synthesis. Central to this hypothesis concerning such interactions between neurons and glia is an astrocytic localization of glutamine synthetase (Martinez-Hernandezet al., 1977)coupled with a highaffinity uptake of glutamate by astrocytes (Hertz, 1979). Several studies have been made to characterize this glutamate uptake in glial cells (Balcar et al., 1977; Hertz et al., 1978; Drejer et al., 1982; Ramaharobandro et al., 1982;Brookes, 1988)and cultured glioma cell lines (Henn et al., 1974; Nicklas and Browning, 1983; Waniewski and Martin, 1984). The kinetic parameters, Na' dependence, and substrate specificityof glutamate uptake in the normal astrocyte cultures were consistent
with the commonly occurring system designated XiG (Drejer et al., 1982; Brookes, 1988). System X i G is a Naf-dependent, anionic amino acid transport system especially reactive with aspartate and glutamate (as for the naming of amino acid transport systems, refer to Bannai et al., 1984;Christensen, 1984a,b).In contrast to normal astrocyte cultures, there is evidence for at least two transport systems for glutamate, i.e., Na+dependent and Naf-independent systems, in glioma cells (Henn et al., 1974;Waniewski and Martin, 1984). In the present experiments, we have examined the glutamate uptake in cultured astrocytes and C-6 glioma cells and found that both cells exhibited Na+-dependent and Naf-independent uptake. The Na+-dependentuptake was consistent with system XiG,whereas the Naf-
Received November 17, 1989; revised manuscript received April 20, 1990; accepted June 11, 1990. Address correspondence and reprint requests to Dr. S. Bannai at Department of Biochemistry, Tsukuba University Medical School, Tsukuba, Ibaraki 305, Japan.
Abbreviations used: BCH, 2-aminobicyclo[2.2.I]heptane-2-carboxylic acid; MEM, Eagle's minimum essential medium; PBS, phosphate buffered saline.
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Uptake of Glutamate and Cystine in C-6 Glioma Cells and in Cultured Astrocytes Yukie Cho and Shiro Bannai Department of Biochemistry, Tsukuba University Medical School, Tsukuba, Ibaraki, Japan
Abstract: The uptake of glutamate in rat glioma C-6 cells and cultured astrocytes derived from rat cerebral hemispheres was found to be mediated by a Na+-dependent and a Na+independent system. The Na+-dependent system was inhibited by aspartate and was consistent with the commonly ocThe Na+-independent curring system designated system X ~ .G system was inhibited by cystine and was consistent with system G- described in various types of cells in the periphery. It was also found that quisqualate selectively and competitively interfered with the Na'hdependent glutamate uptake. In C-6 cells, the glutamate uptake via systems XiG and x; accounted for approximately 35% and 55% of the total uptake, respectively, at 0.05 mM glutamate. In cultured astrocytes,
the glutamate uptake via system XiGwas very potent, whereas the uptake via system x i was relatively weak and its contribution to the total uptake of glutamate seemed almost negligible. However, in both C-6 cells and astrocytes, system was necessary for the uptake of cystine, another substrate of system x i . Cystine in the culture medium was an essential precursor of glutathione, and the inhibition of the cystine uptake by excess glutamate as a competitor led to a severe deficiency in glutathione, followed by cell degeneration. Key Words: Glutamate transport-Cystine-Glutathione-C-6 glioma cells-Astrocytes. Cho Y. and Bannai S. Uptake of glutamate and cystine in C-6 glioma cells and in cultured astrocytes. J. Neurochem. 55, 209 1-2097 (1 990).
Glutamate is a primary candidate for a role as an excitatory transmitter, and a glutamine cycle has been proposed as a mechanism for maintaining neuronal glutamate levels in the face of continual loss by transmitter release (Van den Berg and Garfinkel, 1971). According to this cycle, glutamate released from neurons as a transmitter is accumulated by glia and converted to glutamine. The glutamine is then returned to the neurons, where it provides renewed substrate for neurotransmitter synthesis. Central to this hypothesis concerning such interactions between neurons and glia is an astrocytic localization of glutamine synthetase (Martinez-Hernandezet al., 1977)coupled with a highaffinity uptake of glutamate by astrocytes (Hertz, 1979). Several studies have been made to characterize this glutamate uptake in glial cells (Balcar et al., 1977; Hertz et al., 1978; Drejer et al., 1982; Ramaharobandro et al., 1982;Brookes, 1988)and cultured glioma cell lines (Henn et al., 1974; Nicklas and Browning, 1983; Waniewski and Martin, 1984). The kinetic parameters, Na' dependence, and substrate specificityof glutamate uptake in the normal astrocyte cultures were consistent
with the commonly occurring system designated XiG (Drejer et al., 1982; Brookes, 1988). System X i G is a Naf-dependent, anionic amino acid transport system especially reactive with aspartate and glutamate (as for the naming of amino acid transport systems, refer to Bannai et al., 1984;Christensen, 1984a,b).In contrast to normal astrocyte cultures, there is evidence for at least two transport systems for glutamate, i.e., Na+dependent and Naf-independent systems, in glioma cells (Henn et al., 1974;Waniewski and Martin, 1984). In the present experiments, we have examined the glutamate uptake in cultured astrocytes and C-6 glioma cells and found that both cells exhibited Na+-dependent and Naf-independent uptake. The Na+-dependentuptake was consistent with system XiG,whereas the Naf-
Received November 17, 1989; revised manuscript received April 20, 1990; accepted June 11, 1990. Address correspondence and reprint requests to Dr. S. Bannai at Department of Biochemistry, Tsukuba University Medical School, Tsukuba, Ibaraki 305, Japan.
Abbreviations used: BCH, 2-aminobicyclo[2.2.I]heptane-2-carboxylic acid; MEM, Eagle's minimum essential medium; PBS, phosphate buffered saline.
2091
