Functional expression of particular isoforms of excitatory amino acid transporters by rodent cartilage.
Functional expression of particular isoforms of excitatory amino acid transporters by rodent cartilage.
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DOI：
10.1016/j.bcp.2005.04.025
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发表时间：
2005-07
影响因子：
5.8
通讯作者：
E. Hinoi;Liyang Wang;Akihiro Takemori;Y. Yoneda
中科院分区：
文献类型：
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作者：
E. Hinoi;Liyang Wang;Akihiro Takemori;Y. Yoneda
在本研究中，我们试图证明功能性表达的啮齿动物软骨的兴奋性氨基酸转运蛋白（EAAT）的特定亚型基本上需要中央amatergic信号终止。在培养7-21天的大鼠肋软骨细胞中，通过逆转录聚合酶链反应（RT-PCR），首次显示了神经元EAAT亚型兴奋性氨基酸载体1（EAAC 1）以及胶质细胞亚型如谷氨酸天冬氨酸转运体（GLAST）和谷氨酸转运体1（GLT-1）的mRNA组成型表达。Western印迹分析证实了GLAST和GLT-1在培养的软骨细胞中的相应蛋白的表达。[3 H]谷氨酸（Glu）的蓄积以温度和钠依赖性方式发生，其生化和药理学特征与培养7天的软骨细胞中的脑EAAT相似，而[3 H]Glu蓄积由单一组分组成，Km为39.1±2.3μM，Vmax为1320± 120 pmol/mg protein/min。在器官型培养的跖骨分离血管化前从胚胎小鼠，其中细胞经历了成熟的发展，从静止到增殖，prehypertrophic，肥大和钙化的软骨细胞在细胞分化的渐进顺序，此外，mRNA表达被视为GLAST，GLT-1和EAAT 4，但不是EAAC 1亚型。免疫组织化学分析显示，在培养的跖骨和分离的胫骨切片中，GLAST、GLT-1和EAAT 4亚型的分布情况彼此不同。这些结果表明，细胞外的Glu可以清除到细胞内的位置，通过特定的神经胶质和/或神经元EAAT亚型功能表达的啮齿类动物软骨。
In the present study, we have attempted to demonstrate functional expression by the rodent cartilage of particular isoforms of excitatory amino acid transporters (EAATs) essentially required for central glutamatergic signal termination. Constitutive expression of mRNA was shown for the first time with the neuronal EAAT subtype excitatory amino acid carrier-1 (EAAC1), in addition to glial subtypes such as glutamate aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), in rat costal chondrocytes cultured for 7–21 days on reverse transcription polymerase chain reaction (RT-PCR). Western blotting analysis confirmed the expression of corresponding proteins for both GLAST and GLT-1 in cultured chondrocytes. The accumulation of [3H]glutamate (Glu) occurred in a temperature- and sodium-dependent manner with biochemical and pharmacological profiles similar to those seen for brain EAATs in chondrocytes cultured for 7 days, while [3H]Glu accumulation consisted of a single component with a Kmof 39.1±2.3μM and a Vmaxof 1320±120pmol/mg protein/min, respectively. In organotypic cultured metatarsals isolated before vascularization from embryonic mice, where cells underwent maturational development from resting to proliferating, prehypertrophic, hypertrophic and calcified chondrocytes in a progressive order of cellular differentiation, moreover, mRNA expression was seen for GLAST, GLT-1 and EAAT4 but not for EAAC1 subtypes. Immunohistochemical analysis revealed distribution profiles different from each other with GLAST, GLT-1 and EAAT4 isoforms in sections of cultured metatarsals and isolated tibiae. These results suggest that extracellular Glu could be cleared up into intracellular locations through particular glial and/or neuronal EAAT isoforms functionally expressed by the rodent cartilage.