Susarla Bala T S, Seal Rebecca P, Zelenaia Olga, Watson Deborah J, Wolfe John H, Amara Susan G, Robinson Michael B
Department of Pediatrics, University of Pennsylvania and Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
J Neurochem. 2004 Dec;91(5):1151-63. doi: 10.1111/j.1471-4159.2004.02791.x.
Many neurotransmitter transporters, including the GLT-1 and EAAC1 subtypes of the glutamate transporter, are regulated by protein kinase C (PKC) and these effects are associated with changes in cell surface expression. In the present study, the effects of PKC activation on the glutamate aspartate transporter (GLAST) subtype of glutamate transporter were examined in primary astrocyte cultures. Acute (30 min) exposure to the phorbol 12-myristate 13-acetate (PMA) increased (approximately 20%) transport activity but had the opposite effect on both total and cell surface immunoreactivity. Chronic treatment (6 or 24 h) with PMA had no effect on transport activity but caused an even larger decrease in total and cell surface immunoreactivity. This loss of immunoreactivity was observed using antibodies directed against three different cytoplasmic epitopes, and was blocked by the PKC antagonist, bisindolylmaleimide II. We provide biochemical and pharmacological evidence that the activity observed after treatment with PMA is mediated by GLAST. Two different flag-tagged variants of the human homolog of GLAST were introduced into astrocytes using lentiviral vectors. Although treatment with PMA caused a loss of transporter immunoreactivity, flag immunoreactivity did not change in amount or size. Together, these studies suggest that activation of PKC acutely up-regulates GLAST activity, but also results in modification of several different intracellular epitopes so that they are no longer recognized by anti-GLAST antibodies. We found that exposure of primary cultures of neurons/astrocytes to transient hypoxia/glucose deprivation also caused a loss of GLAST immunoreactivity that was attenuated by the PKC antagonist, bisindolylmaleimide II, suggesting that some acute insults previously thought to cause a loss of GLAST protein may mimic the phenomenon observed in the present study.
许多神经递质转运体，包括谷氨酸转运体的GLT-1和EAAC1亚型，都受蛋白激酶C（PKC）调节，这些效应与细胞表面表达的变化有关。在本研究中，我们在原代星形胶质细胞培养物中检测了PKC激活对谷氨酸转运体的谷氨酸-天冬氨酸转运体（GLAST）亚型的影响。急性（30分钟）暴露于佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）可使转运活性增加（约20%），但对总免疫反应性和细胞表面免疫反应性产生相反的影响。用PMA进行慢性处理（6或24小时）对转运活性没有影响，但导致总免疫反应性和细胞表面免疫反应性更大程度的降低。使用针对三种不同胞质表位的抗体观察到这种免疫反应性的丧失，并且被PKC拮抗剂双吲哚马来酰亚胺II阻断。我们提供了生化和药理学证据，证明用PMA处理后观察到的活性是由GLAST介导的。使用慢病毒载体将人源GLAST的两种不同的带有Flag标签的变体引入星形胶质细胞。尽管用PMA处理导致转运体免疫反应性丧失，但Flag免疫反应性在数量或大小上没有变化。总之，这些研究表明，PKC的激活急性上调GLAST活性，但也导致几种不同的细胞内表位发生修饰，使其不再被抗GLAST抗体识别。我们发现，将神经元/星形胶质细胞的原代培养物暴露于短暂缺氧/葡萄糖剥夺也会导致GLAST免疫反应性丧失，而PKC拮抗剂双吲哚马来酰亚胺II可减弱这种丧失，这表明一些先前认为会导致GLAST蛋白丧失的急性损伤可能模拟了本研究中观察到的现象。
