Characterization of Na+-coupled glutamate/aspartate transport by a rat brain astrocyte line expressing GLAST and EAAC1.
Characterization of Na+-coupled glutamate/aspartate transport by a rat brain astrocyte line expressing GLAST and EAAC1.
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表达 GLAST 和 EAAC1 的大鼠脑星形胶质细胞系的 Na 偶联谷氨酸/天冬氨酸转运的表征。
DOI：
10.1007/s00232-001-0025-1
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Randles,J
中科院分区：
文献类型：
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作者：
Kimmich,GA;Roussie,J;Manglapus,M;Randles,J
研究了长期培养的大鼠脑星形胶质细胞（RBA）悬浮液对d-天冬氨酸（d-Asp）的摄取，作为表征细胞中谷氨酸/天冬氨酸（Glu/Asp）转运体亚型功能和调节的一种手段。d-Asp内流具有Na+依赖性，Km = 5 μmandVmax= 0.7 nmoles · min-1· mg protein-1。入渗量f[Na+]呈S形，Na +Km = 12 μ m，Hill系数为1.9。细胞建立稳态-Asp梯度> 3，000倍。佛波酯（PMA）可增强细胞对D-Asp的摄取，使Vmax增加2倍，达到近6,000倍的梯度，而Km无变化，在初始[d-Asp] = 10 μm时，RBA占总d-Asp的90%以上，细胞外水平降至1 μm以下。耗散ΔμNa+的离子载体抑制梯度形成。Genistein（GEN，100 μm）是一种PTK抑制剂，可使ind-Asp降低40%。PMA（4α-PMA）和GEN（大豆苷元）的非活性类似物没有可检测的作用，尽管当GEN存在时仍然发生刺激性PMA反应。PMA对Na+-偶联的ALA摄取没有影响，但GEN具有刺激作用，这一事实进一步表明了作用的特异性。丝氨酸-O-硫酸盐（S-O-S）、苏式羟基-天冬氨酸（THA）、l-Asp和l-Glu强烈抑制d-Asp摄取，但d-Glu、红藻氨酸（KA）或二氢红藻氨酸（DHK）不抑制d-Asp摄取，这是GLAST和EAAC 1转运蛋白亚型的特征性抑制模式。通过RT-PCR检测两种亚型的mRNA，并且用适当的抗体进行的Western印迹显示这两种蛋白质在这些细胞中表达。
d-Aspartate (d-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells.d-Asp influx is Na+-dependent withKm= 5 μmandVmax= 0.7 nmoles · min−1· mg protein−1. Influx is sigmoidal as f[Na+] withNa+Km∼ 12 μmand Hill coefficient of 1.9. The cells establish steady-stated-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase inVmax, with no change inKm.At initial [d-Asp] = 10 μm, RBA take up more than 90% of totald-Asp, and extracellular levels are reduced to levels below 1 μm. Ionophores that dissipate the ΔμNa+inhibit gradient formation. Genistein (GEN, 100 μm), a PTK inhibitor, causes a 40% decrease ind-Asp. Inactive analogs of PMA (4α-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na+-coupled ALA uptake, but GEN is stimulatory.d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA),l-Asp, andl-Glu, but not byd-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells.