Uptake Study in Lysosome-Enriched Fraction: Critical Involvement of Lysosomal Trapping in Quinacrine Uptake but Not Fluorescence-Labeled Verapamil Transport at Blood-Retinal Barrier.

Yoshiyuki Kubo,Saki Konakawa,Shin-Ichi Akanuma,Miki Yamada,Ken-Ichi Hosoya

doi:10.3390/pharmaceutics12080747

Abstract

Lysosomal trapping at the blood–retinal barrier (BRB) was investigated through quinacrine and fluorescence-labeled verapamil (EFV) uptake. Quinacrine uptake by conditionally immortalized rat retinal capillary endothelial (TR-iBRB2) cells suggested saturable and non-saturable transport processes in the inner BRB. The reduction of quinacrine uptake by bafilomycin A1 suggested quinacrine distribution to the acidic intracellular compartments of the inner BRB, and this notion was also supported in confocal microscopy. In the study using the lysosome-enriched fraction of TR-iBRB2 cells, quinacrine uptake was inhibited by bafilomycin A1, suggesting the lysosomal trapping of quinacrine in the inner BRB. Pyrilamine, clonidine, and nicotine had no effect on quinacrine uptake, suggesting the minor role of lysosomal trapping in their transport across the inner BRB. Bafilomycin A1 had no effect on EFV uptake, and lysosomal trapping driven by the acidic interior pH was suggested as a minor mechanism for EFV transport in the inner BRB. The minor contribution of lysosomal trapping was supported by the difference in inhibitory profiles between EFV and quinacrine uptakes. Similar findings were observed in the outer BRB study with the fraction of conditionally immortalized rat retinal pigment epithelial (RPE-J) cells. These results suggest the usefulness of lysosome-enriched fractions in studying lysosomal trapping at the BRB.

Highlights

The blood–retinal barrier (BRB) has two barrier structures, the inner and outer BRB, separating the neural retina and circulating blood [1,2,3]
In TR-iBRB2 cells, quinacrine uptake was significantly reduced by 45% at 4 ◦C (Figure 1A), and significantly increased by 31% at pH 8.4, while the uptake exhibited no significant change in K+- or Li+-replacement buffer (Figure 1B)
The cellular uptake study suggests that quinacrine was distributed to the acidic intracellular compartments of the inner BRB, and this was supported by the confocal microscopy results

Summary

Introduction

The blood–retinal barrier (BRB) has two barrier structures, the inner and outer BRB, separating the neural retina and circulating blood [1,2,3]. Previous progress in the study of the BRB achieved using in vivo and in vitro methods has demonstrated the critical roles of blood-to-retina transport across the BRB in the retinal homeostasis [2,3]. These studies have clearly shown that nutrient transport at the BRB involves various membrane transporters, such as glucose transporter (GLUT1/SLC2A1), taurine transporter (TAUT/SLC6A6), creatine transporter (CRT/SLC6A8), cationic amino acid transporter (CAT1/SLC7A1), equilibrative nucleoside transporter (ENT2/SLC29A2), and riboflavin transporter (RFVTs/SLC52A) [3,4,5,6,7,8,9,10,11]

Methods

Results

Discussion

Conclusion