Blood-to-Retina Transport of Fluorescence-Labeled Verapamil at the Blood-Retinal Barrier
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 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.
Our findings provide visual evidence to support the significance of carrier-mediated transport in the blood-to-retina verapamil transport at the BRB.
Key wordsblood-retinal barrier lysosomal trapping neuroprotectants organic cation transport verapamil
ATP-biding cassette transporter subfamily B member 1
ATP-biding cassette transporter subfamily C member 1
ATP-biding cassette transporter subfamily C member 4
ATP-biding cassette transporter subfamily G member 2
Association for Research in Vision and Ophthalmology
Breast cancer resistance protein
- C/M ratio
EverFluor FL Verapamil (BODIPY® FL Verapamil)
- FI ratio
Fluorescence intensity ratio
Ganglion cell layer
50% Inhibitory concentration
Inner limiting membrane
Inner nuclear layer
Inner plexiform layer
Multi drug resistance 1
Multi drug resistance-related protein 1
Multi drug resistance-related protein 4
Organic anion transporter 3
Organic anion transporting polypeptide 1a4
Outer limiting membrane
Outer nuclear layer
Outer plexiform layer
Photoreceptor outer segment
Rhodamine B isothiocyanate-dextran
Retinal pigment epithelium
Retinal uptake index
Solute carrier organic anion
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.
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