Redzic Zoran B, Biringer Jean, Barnes Kay, Baldwin Stephen A, Al-Sarraf Hameed, Nicola Pieris A, Young James D, Cass Carol E, Barrand Margery A, Hladky Stephen B
Department of Pharmacology, University of Cambridge, UK.
J Neurochem. 2005 Sep;94(5):1420-6. doi: 10.1111/j.1471-4159.2005.03312.x.
This study investigated mRNA expression and protein localization of equilibrative and concentrative nucleoside transporters (ENTs, CNTs) in primary cultures of rat brain endothelial cells (RBEC) and rat choroid plexus epithelial cells (RCPEC). Reverse transcriptase PCR analysis revealed that RBEC and RCPEC contained mRNA for rENT1, rENT2 and rCNT2 and for rENT1, rENT2, rCNT2 and rCNT3, respectively. Immunoblotting of membrane fractions of RBEC, fresh RCPEC and primary cultures of RCPEC revealed the presence of rENT1, rENT2 and rCNT2 proteins in all samples. Measurement of [14C]adenosine uptake into cells grown as monolayers on permeable plastic supports revealed a polarized distribution of Na+-dependent adenosine uptake in that CNT activity was associated exclusively in membranes of RBEC facing the lower chamber (which corresponds to the surface facing the interstitial fluid in situ) and in membranes of RCPEC facing the upper chamber (which corresponds to the surface facing the cerebrospinal fluid in situ). In both RBEC and RCPEC, adenosine uptake from the opposite chambers was Na+-independent and partially inhibited by nitrobenzylthioinosine, indicating the presence of the equilibrative sensitive transporter rENT1.
本研究调查了大鼠脑内皮细胞（RBEC）和大鼠脉络丛上皮细胞（RCPEC）原代培养物中平衡型和浓缩型核苷转运体（ENTs、CNTs）的mRNA表达及蛋白定位。逆转录聚合酶链反应分析显示，RBEC和RCPEC分别含有rENT1、rENT2和rCNT2以及rENT1、rENT2、rCNT2和rCNT3的mRNA。对RBEC、新鲜RCPEC和RCPEC原代培养物的膜组分进行免疫印迹分析，结果显示所有样品中均存在rENT1、rENT2和rCNT2蛋白。对生长在可渗透塑料支持物上的单层细胞进行[14C]腺苷摄取测量，结果显示Na+依赖性腺苷摄取存在极化分布，即CNT活性仅与RBEC面向下腔室的膜（对应于原位面向间质液的表面）以及RCPEC面向上腔室的膜（对应于原位面向脑脊液的表面）相关。在RBEC和RCPEC中，来自相对腔室的腺苷摄取均不依赖于Na+，并被硝基苄硫基肌苷部分抑制，这表明存在平衡型敏感转运体rENT1。
