Rapid stimulation of EAAC1-mediated Na+-dependent L-glutamate transport activity in C6 glioma cells by phorbol ester
Rapid stimulation of EAAC1-mediated Na+-dependent L-glutamate transport activity in C6 glioma cells by phorbol ester
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DOI：
10.1046/j.1471-4159.1996.67020508.x
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发表时间：
1996-08-01
影响因子：
4.7
通讯作者：
Robinson, MB
中科院分区：
文献类型：
--
作者：
Dowd, LA;Robinson, MB
以C6胶质瘤细胞为模型系统，研究EAAC1对Na+依赖的L[H-3]谷氨酸转运的调节作用。佛波醇12-肉豆蔻酸酯(PMA)可使转运活性增加2~3倍，佛波酯可使EAAC1介导的L[H-3]谷氨酸转运活性增加2~3倍，且呈时间和浓度依赖性。PMA预先孵育2分钟就足以使转运活性增加一倍以上，而蛋白质合成抑制剂放线菌酮对此无影响，提示这种增加不依赖于蛋白质合成，PMA刺激转运活性的EC(50)值为80 nM。动力学分析表明，转运活性的增加是由于V-max增加了2.5倍，而K-m没有变化。PMA也同样增加了非代谢类似物D-[H-3]天冬氨酸的转运。然而，在平行试验中，PMA并没有增加C6胶质瘤中依赖于Na+的甘氨酸转运活性。灭活佛波醇酯4α佛波醇12，13-十二酸不能刺激L[H-3]谷氨酸转运，蛋白激酶C抑制剂白屈菜红碱可阻断PMA的刺激作用。磷酸酶抑制剂冈田酸和环孢素A对PMA引起的转运活性的刺激没有影响。钙离子载体A23187对PMA的刺激没有协同作用。在以往的研究中，PMA引起C6胶质瘤对阿米洛利敏感的Na+/H+转运活性迅速增加，而在本研究中，预孵育和与阿米洛利共同孵育对PMA引起的转运活性的刺激没有影响。这些研究表明，蛋白激酶C的激活导致EAAC1介导的转运活性迅速增加。这种钠依赖的L[H-3]谷氨酸转运活性的快速增加可能为中枢神经系统的急性损伤提供了一种新的保护机制。
C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na+-dependent L-[H-3]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold, PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated L-[H-3]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in 'transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase, These data suggest that this increase is independent of protein synthesis, The EC(50) value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in V-max with no change in K-m. PMA also increased the transport of the non metabolizable analogue, D-[H-3] aspartate to the same extent. In parallel assays, PMA did not, however, increase Na+-dependent glycine transport activity in C6 glioma. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate, did not stimulate L-[H-3]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca2+ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na+/H+ transport activity in C6 glioma, In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na+-dependent L-[H-3]glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS.