Blood-to-Retina Transport of Fluorescence-Labeled Verapamil at the Blood-Retinal Barrier
Blood-to-Retina Transport of Fluorescence-Labeled Verapamil at the Blood-Retinal Barrier
复制标题
荧光标记维拉帕米在血视网膜屏障处的血液至视网膜转运
DOI：
10.1007/s11095-018-2384-7
复制
发表时间：
2018
影响因子：
3.7
通讯作者：
Hosoya Ken-ichi
中科院分区：
文献类型：
--
作者：
Kubo Yoshiyuki;Nakazawa Ayumi;Akanuma Shin-ichi;Hosoya Ken-ichi
目的研究维拉帕米在血-视网膜屏障(BRB)的血到视网膜的转运。方法采用EverFluor FL Verapamil(EFV)作为维拉帕米的荧光探针，采用大鼠颈总动脉灌注的方法观察其跨BRB的转运。结果在视网膜组织中检测到EFV信号时，视网膜组织中检测到EFV信号。在tr-iBRB2细胞中，EFV的定位与溶酶促进剂LysoTracker®Red不同，并且不会因NH4Cl急性处理而改变。在RPE-J细胞中，可部分观察到EFV的点状分布，这种分布可被NH4Cl急性处理后减少。Tr-iBRB2细胞对EFV的摄取是温度依赖的，膜电位和pH不依赖的，NH4Cl处理可显著降低EFV摄取，而不同的胞外pH和V-ATPase抑制剂对EFV摄取无明显影响。RT-iBRB2细胞对EFV的摄取被阳离子药物抑制，并被维拉帕米以浓度依赖的方式抑制，IC50为98.0μM。结论我们的研究结果为载体介导的维拉帕米在血到视网膜的转运中的重要性提供了视觉证据。
PurposeTo investigate the blood-to-retina verapamil transport at the blood-retinal barrier (BRB).MethodsEverFluor FL Verapamil (EFV) was adopted as the fluorescent probe of verapamil, and its transport across the BRB was investigated with common carotid artery infusion in rats. EFV transport at the inner and outer BRB was investigated with TR-iBRB2 cells and RPE-J cells, respectively.ResultsThe signal of EFV was detected in the retinal tissue during the weak signal of cell impermeable compound. In TR-iBRB2 cells, the localization of EFV differed from that of LysoTracker®Red, a lysosomotropic agent, and was not altered by acute treatment with NH4Cl. In RPE-J cells, the punctate distribution of EFV was partially observed, and this was reduced by acute treatment with NH4Cl. EFV uptake by TR-iBRB2 cells was temperature-dependent and membrane potential- and pH-independent, and was significantly reduced by NH4Cl treatment during no significant effect obtained by different extracellular pH and V-ATPase inhibitor. The EFV uptake by TR-iBRB2 cells was inhibited by cationic drugs, and inhibited by verapamil in a concentration-dependent manner with an IC50of 98.0 μM.ConclusionsOur findings provide visual evidence to support the significance of carrier-mediated transport in the blood-to-retina verapamil transport at the BRB.