Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630, Sugitani, Toyama, 930-0194, Japan.
Pharm Res. 2018 Mar 12;35(5):93. doi: 10.1007/s11095-018-2384-7.
To investigate the blood-to-retina verapamil transport at the blood-retinal barrier (BRB).
EverFluor 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.
The 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 NHCl. In RPE-J cells, the punctate distribution of EFV was partially observed, and this was reduced by acute treatment with NHCl. EFV uptake by TR-iBRB2 cells was temperature-dependent and membrane potential- and pH-independent, and was significantly reduced by NHCl 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 IC of 98.0 μM.
Our findings provide visual evidence to support the significance of carrier-mediated transport in the blood-to-retina verapamil transport at the BRB.
研究血视网膜屏障（BRB）中血液到视网膜的维拉帕米转运。
采用EverFluor FL 维拉帕米（EFV）作为维拉帕米的荧光探针，通过大鼠颈总动脉输注研究其穿过 BRB 的转运情况。采用 TR-iBRB2 细胞和 RPE-J 细胞分别研究 EFV 在内外 BRB 的转运。
在细胞不可渗透化合物的弱信号期间，在视网膜组织中检测到 EFV 的信号。在 TR-iBRB2 细胞中，EFV 的定位与溶酶体靶向剂 LysoTracker Red 不同，并且不受 NHCl 的急性处理改变。在 RPE-J 细胞中，EFV 的点状分布部分被观察到，并且该分布在 NHCl 的急性处理下减少。TR-iBRB2 细胞对 EFV 的摄取依赖于温度，与膜电位和 pH 无关，并且在没有明显影响的情况下，NHCl 处理显著降低，而不同的细胞外 pH 和 V-ATPase 抑制剂则没有明显影响。TR-iBRB2 细胞对 EFV 的摄取被阳离子药物抑制，并且被维拉帕米以浓度依赖性方式抑制，IC 为 98.0 μM。
我们的发现提供了视觉证据，支持载体介导的转运在 BRB 中血液到视网膜的维拉帕米转运中的重要性。
