Purine receptors and Ca2+ signalling in the human blood-brain barrier endothelial cell line hCMEC/D3
Purine receptors and Ca2+ signalling in the human blood-brain barrier endothelial cell line hCMEC/D3
复制标题
DOI：
10.1007/s11302-011-9262-7
复制
发表时间：
2012-03-01
影响因子：
3.5
通讯作者：
Ngezahayo, Anaclet
中科院分区：
文献类型：
--
作者：
Bintig, Willem;Begandt, Daniela;Ngezahayo, Anaclet
应用分子生物学、基因沉默和钙离子成像技术对人血脑屏障内皮细胞（hCMEC/D3）嘌呤受体的表达和生理特性进行了研究。逆转录聚合酶链反应显示G蛋白偶联受体P2 Y（2）-、P2 Y（6）-、P2 Y（11）-以及离子型P2 X（4）-、P2 X（5）-和P2 X（7）-受体的表达。Fura-2比率法显示，在单细胞中，三磷酸腺苷（ATP）或三磷酸尿苷（UTP）介导了细胞内Ca 2+浓度（[Ca 2 +]（i））从150 nM至300 nM的变化。[Ca 2 +]（i）的变化对应于Fluo-4的荧光强度增加四倍至五倍，Fluo-4用于高通量实验。使用不同激动剂[UTP γ S、ATP γ S、尿苷二磷酸（UDP）、腺苷二磷酸（ADP）、BzATP、α β-meATP]和拮抗剂的药理学研究（MRS 2578或NF 340）以及细胞内介质的抑制剂（U 73122和2-APB）显示PLC-IP 3级联介导的Ca 2+释放，表明核苷酸诱导的Ca 2+信号主要与P2 Y有关（2、6和11）受体。P2 Y（2）受体的基因沉默降低ATP或UTP诱导的Ca 2+信号，并抑制由P2 Y（6）和P2 Y（11）更特异性的激动剂如UDP（P2 Y（6））、BzATP（P2 Y（11））和ATP γ S（P2 Y（11））介导的Ca 2+信号。本报告鉴定了P2 Y（2）受体亚型作为参与hCMEC/D3细胞Ca 2+信号传导的主要嘌呤受体。
The expression and physiology of purine receptors of the human blood-brain barrier endothelial cells were characterised by application of molecular biological, gene-silencing and Ca2+-imaging techniques to hCMEC/D3 cells. Reverse transcription polymerase chain reaction showed the expression of the G-protein-coupled receptors P2Y(2)-, P2Y(6)-, P2Y(11)- as well as the ionotropic P2X(4)-, P2X(5)- and P2X(7)-receptors. Fura-2 ratiometry revealed that adenosine triphosphate (ATP) or uridine triphosphate (UTP) mediated a change in the intracellular Ca2+ concentration ([Ca2+](i)) from 150 to 300 nM in single cells. The change in [Ca2+](i) corresponded to a fourfold to fivefold increase in the fluorescence intensity of Fluo-4, which was used for high-throughput experiments. Pharmacological dissection using different agonists [UTP gamma S, ATP gamma S, uridine diphosphate (UDP), adenosine diphosphate (ADP), BzATP, alpha beta-meATP] and antagonist (MRS2578 or NF340) as well as inhibitors of intracellular mediators (U73122 and 2-APB) showed a PLC-IP3 cascade-mediated Ca2+ release, indicating that the nucleotide-induced Ca2+ signal was mainly related to P2Y(2, 6 and 11) receptors. The gene silencing of the P2Y(2) receptor reduced the ATP- or UTP-induced Ca2+ signal and suppressed the Ca2+ signal mediated by P2Y(6) and P2Y(11) more specific agonists like UDP (P2Y(6)), BzATP (P2Y(11)) and ATP gamma S (P2Y(11)). This report identifies the P2Y(2) receptor subtype as the main purine receptor involved in Ca2+ signalling of the hCMEC/D3 cells.