GLUTAMATE EFFLUX VIA THE REVERSAL OF THE SODIUM-DEPENDENT GLUTAMATE TRANSPORTER CAUSED BY GLYCOLYTIC INHIBITION IN RAT CULTURED ASTROCYTES
GLUTAMATE EFFLUX VIA THE REVERSAL OF THE SODIUM-DEPENDENT GLUTAMATE TRANSPORTER CAUSED BY GLYCOLYTIC INHIBITION IN RAT CULTURED ASTROCYTES
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DOI：
10.1016/0306-4522(94)90523-1
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发表时间：
1994-12-01
期刊：
影响因子：
3.3
通讯作者：
NINOMIYA, M
中科院分区：
文献类型：
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作者：
GEMBA, T;OSHIMA, T;NINOMIYA, M
[H-3]用1 mM的糖酵解抑制剂碘乙酸预先孵育30分钟，可完全降低培养的大鼠星形胶质细胞对[H-3]谷氨酸的摄取。这种处理显著降低了培养的星形胶质细胞的能荷势，但没有造成细胞膜的破坏。为研究碘乙酸对星形胶质细胞谷氨酸释放的影响，在谷氨酰胺合成酶抑制剂蛋氨酸亚硫胺存在下，将[H-3]L-谷氨酸预加到星形胶质细胞中，并测定1 mM碘乙酸作用30min后星形胶质细胞内总放射性的变化，与未处理的星形胶质细胞进行比较。在处理过程中，大约40%的细胞内谷氨酸被排出。降低细胞外钾离子浓度可减少细胞外钾离子外流。用钠离子敏感的荧光探针(钠结合的苯并呋喃异磷酸盐)测量细胞内钠离子浓度，当加入1 mM的碘乙酸时，细胞内钠浓度逐渐增加到30 mM。这些结果表明，在糖酵解抑制过程中，细胞内Na+依赖的转运体发生逆转，导致谷氨酸外流，这可能是由于细胞内Na+超载所致。选择性Na+-H+逆向转运蛋白抑制剂1 mU M5-[N-乙基-N-异丙基]阿米洛利可完全减轻碘乙酸引起的Na+超载，但这并不能阻止碘乙酸诱导的谷氨酸外流。用pH敏感的荧光探针[2‘，7’-bis(carboxyethyl)-5，6-carboxy-fluorescein]，测得的细胞内pH经碘乙酸处理后逐渐降低到7.1.另一方面，在5-[N-乙基-N-异丙基]胺存在下，碘乙酸引起的细胞内酸中毒更为迅速和严重。这些结果表明，依赖Na+的谷氨酸转运体的逆转可能不仅是由细胞内Na+超载引起的，也可能是由细胞内酸中毒引起的。
[H-3]L-Glutamate uptake in cultured rat astrocytes was completely reduced by 30 min preincubation with 1 mM of iodoacetic acid, a glycolytic inhibitor. This treatment significantly reduced the energy charge potential, but did not cause membrane destruction in the cultured astrocytes. To examine the effect of iodoacetic acid on the glutamate release, [H-3]L-glutamate was preloaded into astrocytes in the presence of methionine sulfoximine, a glutamine synthetase inhibitor, and the total intracellular radioactivity was measured after 30-min treatment with 1 mM iodoacetic acid for comparison with non-treated astrocytes. During the treatment, about 40% of the total intracellular glutamate content was effluxed. This efflux could be decreased by reducing the extracellular potassium ion concentration. The intracellular sodium concentration, measured with a sodium ion-sensitive fluorescent probe (sodium-binding benzofuran isophtalate), gradually increased to 30 mM on addition of 1 mM iodoacetic acid. These results indicate that the glutamate efflux via reversal of the Na+-dependent transporter occurred during glycolytic inhibition, and which may be caused by intracellular Na+ overload. Such an iodoacetic acid-induced Na+ overload could be completely diminished by pretreatment with 1 mu M 5-[N-ethyl-N -isopropyl]amiloride, a selective Na+-H+ antiporter inhibitor, but even this did not stop the iodoacetic acid-induced glutamate efflux. The intracellular pH, measured by a pH-sensitive fluorescent probe [2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein], was gradually decreased to 7.1 by the iodoacetic acid treatment. On the other hand, iodoacetic acid-induced intracellular acidosis was more rapid and severe in the presence of 5-[N-ethyl-N-isopropyl]amiloride.These results suggest that the reversal of the Na+-dependent glutamate transporter may be caused by not only intracellular Na+ overload but also intracellular acidosis.