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Blood-to-Retina Transport of Fluorescence-Labeled Verapamil at the Blood-Retinal Barrier

  • Yoshiyuki Kubo
  • Ayumi Nakazawa
  • Shin-ichi Akanuma
  • Ken-ichi Hosoya
Research Paper

Abstract

Purpose

To investigate the blood-to-retina verapamil transport at the blood-retinal barrier (BRB).

Methods

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.

Results

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 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 IC50 of 98.0 μM.

Conclusions

Our findings provide visual evidence to support the significance of carrier-mediated transport in the blood-to-retina verapamil transport at the BRB.

Key words

blood-retinal barrier lysosomal trapping neuroprotectants organic cation transport verapamil 

Abbreviations

ABCB1

ATP-biding cassette transporter subfamily B member 1

ABCC1

ATP-biding cassette transporter subfamily C member 1

ABCC4

ATP-biding cassette transporter subfamily C member 4

ABCG2

ATP-biding cassette transporter subfamily G member 2

ARVO

Association for Research in Vision and Ophthalmology

BCRP

Breast cancer resistance protein

BRB

Blood-retinal barrier

CH

Choroid

C/M ratio

Cell-to-medium ratio

CNG

Cyclic nucleotide-gated

DAPI

4′,6-Diamidino-2-phenylindole

ECF

Extracellular fluid

EFV

EverFluor FL Verapamil (BODIPY® FL Verapamil)

FCCP

Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone

FI ratio

Fluorescence intensity ratio

GCL

Ganglion cell layer

IC50

50% Inhibitory concentration

ILM

Inner limiting membrane

INL

Inner nuclear layer

IPL

Inner plexiform layer

Km

Michaelis constant

LTR

LysoTracker® Red

MDR1

Multi drug resistance 1

MPP+

1-Methyl-4-phenylpyridinium

MRP1

Multi drug resistance-related protein 1

MRP4

Multi drug resistance-related protein 4

Oat3

Organic anion transporter 3

Oatp1a4

Organic anion transporting polypeptide 1a4

OLM

Outer limiting membrane

ONL

Outer nuclear layer

OPL

Outer plexiform layer

PAH

p-Aminohippuric acid

PBS

Phosphate-buffered saline

P-gp

P-glycoprotein

POS

Photoreceptor outer segment

Rho-D

Rhodamine B isothiocyanate-dextran

RPE

Retinal pigment epithelium

RUI

Retinal uptake index

SLC

Solute carrier

SLCO

Solute carrier organic anion

TEA

Tetraethylammonium

V-ATPase

Vacuolar-type H+-ATPase

Notes

Acknowledgments and Disclosures

The present study was financially supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant number JP16H05110 and JP17K08409), JSPS Core-to-Core Program (B. Asia-Africa Science Platforms), and Research Grants from the Smoking Research Foundation and the Takeda Science Foundation. The authors thank Miss Reina Makino for her technical support involving confocal microscopy. The authors declare no conflict of interest.

Supplementary material

11095_2018_2384_MOESM1_ESM.doc (44 kb)
ESM 1 (DOC 43 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Yoshiyuki Kubo
    • 1
  • Ayumi Nakazawa
    • 1
  • Shin-ichi Akanuma
    • 1
  • Ken-ichi Hosoya
    • 1
  1. 1.Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan

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