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



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 words

blood-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


Blood-retinal barrier



C/M ratio

Cell-to-medium ratio


Cyclic nucleotide-gated




Extracellular fluid


EverFluor FL Verapamil (BODIPY® FL Verapamil)


Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone

FI ratio

Fluorescence intensity ratio


Ganglion cell layer


50% Inhibitory concentration


Inner limiting membrane


Inner nuclear layer


Inner plexiform layer


Michaelis constant


LysoTracker® Red


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


p-Aminohippuric acid


Phosphate-buffered saline




Photoreceptor outer segment


Rhodamine B isothiocyanate-dextran


Retinal pigment epithelium


Retinal uptake index


Solute carrier


Solute carrier organic anion




Vacuolar-type H+-ATPase


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)


  1. 1.
    Chao HM, Chidlow G, Melena J, Wood JP, Osborne NN. An investigation into the potential mechanisms underlying the neuroprotective effect of clonidine in the retina. Brain Res. 2000;877:47–57.CrossRefPubMedGoogle Scholar
  2. 2.
    Ristori C, Filippi L, Dal Monte M, Martini D, Cammalleri M, Fortunato P, et al. Role of the adrenergic system in a mouse model of oxygen-induced retinopathy: antiangiogenic effects of beta-adrenoreceptor blockade. Invest Ophthalmol Vis Sci. 2011;52:155–70.CrossRefPubMedGoogle Scholar
  3. 3.
    Hosoya K, Tomi M, Tachikawa M. Strategies for therapy of retinal diseases using systemic drug delivery: relevance of transporters at the blood-retinal barrier. Expert Opin Drug Deliv. 2011;8:1571–87.CrossRefPubMedGoogle Scholar
  4. 4.
    Cunha-Vaz JG. The blood-retinal barriers system. Basic concepts and clinical evaluation. Exp Eye Res. 2004;78:715–21.CrossRefPubMedGoogle Scholar
  5. 5.
    Stewart PA, Tuor UI. Blood-eye barriers in the rat: correlation of ultrastructure with function. J Comp Neurol. 1994;340:566–76.CrossRefPubMedGoogle Scholar
  6. 6.
    Hosoya K, Tomi M, Ohtsuki S, Takanaga H, Ueda M, Yanai N, et al. Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat. Exp Eye Res. 2001;72:163–72.CrossRefPubMedGoogle Scholar
  7. 7.
    Hosoya K, Tomi M. Advances in the cell biology of transport via the inner blood-retinal barrier: establishment of cell lines and transport functions. Biol Pharm Bull. 2005;28:1–8.CrossRefPubMedGoogle Scholar
  8. 8.
    Kubo Y, Obata A, Akanuma S, Hosoya K. Impact of cationic amino acid transporter 1 on blood-retinal barrier transport of L-ornithine. Invest Ophthalmol Vis Sci. 2015;56:5925–32.CrossRefPubMedGoogle Scholar
  9. 9.
    Kubo Y, Yahata S, Miki S, Akanuma SI, Hosoya K. Blood-to-retina transport of riboflavin via RFVTs at the inner blood-retinal barrier. Drug Metab Pharmacokinet. 2017;32:92–9.CrossRefPubMedGoogle Scholar
  10. 10.
    Kubo Y, Akanuma S, Hosoya K. Influx transport of cationic drug at the blood-retinal barrier: impact on the retinal delivery of neuroprotectants. Biol Pharm Bull. 2017;40:1139–45.CrossRefPubMedGoogle Scholar
  11. 11.
    Hosoya K, Yamamoto A, Akanuma S, Tachikawa M. Lipophilicity and transporter influence on blood-retinal barrier permeability: a comparison with blood-brain barrier permeability. Pharm Res. 2010;27:2715–24.CrossRefPubMedGoogle Scholar
  12. 12.
    Kubo Y, Fukui E, Akanuma S, Tachikawa M, Hosoya K. Application of membrane permeability evaluated in in vitro analyses to estimate blood-retinal barrier permeability. J Pharm Sci. 2012;101:2596–605.CrossRefPubMedGoogle Scholar
  13. 13.
    Kubo Y, Kusagawa Y, Tachikawa M, Akanuma S, Hosoya K. Involvement of a novel organic cation transporter in verapamil transport across the inner blood-retinal barrier. Pharm Res. 2013;30:847–56.CrossRefPubMedGoogle Scholar
  14. 14.
    Fujii S, Setoguchi C, Kawazu K, Hosoya K. Impact of P-glycoprotein on blood-retinal barrier permeability: comparison of blood-aqueous humor and blood-brain barrier using mdr1a knockout rats. Invest Ophthalmol Vis Sci. 2014;55:4650–8.CrossRefPubMedGoogle Scholar
  15. 15.
    Kubo Y, Shimizu Y, Kusagawa Y, Akanuma S, Hosoya K. Propranolol transport across the inner blood-retinal barrier: potential involvement of a novel organic cation transporter. J Pharm Sci. 2013;102:3332–42.CrossRefPubMedGoogle Scholar
  16. 16.
    Kubo Y, Tsuchiyama A, Shimizu Y, Akanuma S, Hosoya K. Involvement of carrier-mediated transport in the retinal uptake of clonidine at the inner blood-retinal barrier. Mol Pharm. 2014;11:3747–53.CrossRefPubMedGoogle Scholar
  17. 17.
    Tega Y, Kubo Y, Yuzurihara C, Akanuma S, Hosoya K. Carrier-mediated transport of nicotine across the inner blood-retinal barrier: involvement of a novel organic cation transporter driven by an outward H(+) gradient. J Pharm Sci. 2015;104:3069–75.CrossRefPubMedGoogle Scholar
  18. 18.
    Chapy H, Saubaméa B, Tournier N, Bourasset F, Behar-Cohen F, Declèves X, et al. Blood-brain and retinal barriers show dissimilar ABC transporter impacts and concealed effect of P-glycoprotein on a novel verapamil influx carrier. Br J Pharmacol. 2016;173:497–510.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    de Duve C, de Barsy T, Poole B, Trouet A, Tulkens P, Van Hoof F. Commentary. Lysosomotropic agents. Biochem Pharmacol. 1974;23:2495–531.CrossRefPubMedGoogle Scholar
  20. 20.
    Gong Y, Zhao Z, McConn DJ, Beaudet B, Tallman M, Speake JD, et al. Lysosomes contribute to anomalous pharmacokinetic behavior of melanocortin-4 receptor agonists. Pharm Res. 2007;24:1138–44.CrossRefPubMedGoogle Scholar
  21. 21.
    Nadanaciva S, Lu S, Gebhard DF, Jessen BA, Pennie WD, Will Y. A high content screening assay for identifying lysosomotropic compounds. Toxicol in Vitro. 2011;25:715–23.CrossRefPubMedGoogle Scholar
  22. 22.
    Marceau F, Bawolak MT, Lodge R, Bouthillier J, Gagné-Henley A, Gaudreault RC, et al. Cation trapping by cellular acidic compartments: beyond the concept of lysosomotropic drugs. Toxicol Appl Pharmacol. 2012;259:1–12.CrossRefPubMedGoogle Scholar
  23. 23.
    Duvvuri M, Gong Y, Chatterji D, Krise JP. Weak base permeability characteristics influence the intracellular sequestration site in the multidrug-resistant human leukemic cell line HL-60. J Biol Chem. 2004;279:32367–72.CrossRefPubMedGoogle Scholar
  24. 24.
    Kazmi F, Hensley T, Pope C, Funk RS, Loewen GJ, Buckley DB, et al. Lysosomal sequestration (trapping) of lipophilic amine (cationic amphiphilic) drugs in immortalized human hepatocytes (Fa2N-4 cells). Drug Metab Dispos. 2013;41:897–905.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Ohkuma S, Moriyama Y, Takano T. Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence. Proc Natl Acad Sci U S A. 1982;79:2758–62.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Yoshimori T, Yamamoto A, Moriyama Y, Futai M, Tashiro Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem. 1991;266:17707–12.PubMedGoogle Scholar
  27. 27.
    Lemieux B, Percival MD, Falgueyret JP. Quantitation of the lysosomotropic character of cationic amphiphilic drugs using the fluorescent basic amine red DND-99. Anal Biochem. 2004;327:247–51.CrossRefPubMedGoogle Scholar
  28. 28.
    Kubo Y, Seko N, Usui T, Akanuma S, Hosoya K. Lysosomal trapping is present in retinal capillary endothelial cells: insight into its influence on cationic drug transport at the inner blood-retinal barrier. Biol Pharm Bull. 2016;39:1319–24.CrossRefPubMedGoogle Scholar
  29. 29.
    Crivellato E, Candussio L, Rosati AM, Bartoli-Klugmann F, Mallardi F, Decorti G. The fluorescent probe Bodipy-FL-verapamil is a substrate for both P-glycoprotein and multidrug resistance-related protein (MRP)-1. J Histochem Cytochem. 2002;50:731–4.CrossRefPubMedGoogle Scholar
  30. 30.
    Troost J, Lindenmaier H, Haefeli WE, Weiss J. Modulation of cellular cholesterol alters P-glycoprotein activity in multidrug-resistant cells. Mol Pharmacol. 2004;66:1332–9.CrossRefPubMedGoogle Scholar
  31. 31.
    Takasato Y, Rapoport SI, Smith QR. An in situ brain perfusion technique to study cerebrovascular transport in the rat. Am J Phys. 1984;247:H484–93.Google Scholar
  32. 32.
    Yoshida D, Todo H, Hasegawa T, Sugibayashi K. Effect of molecular weight on the dermatopharmacokinetics and systemic disposition of drugs after intracutaneous injection. Eur J Pharm Sci. 2008;35:5–11.CrossRefPubMedGoogle Scholar
  33. 33.
    Egawa G, Nakamizo S, Natsuaki Y, Doi H, Miyachi Y, Kabashima K. Intravital analysis of vascular permeability in mice using two-photon microscopy. Sci Rep. 2013;3:1932.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Puchowicz MA, Xu K, Magness D, Miller C, Lust WD, Kern TS, et al. Comparison of glucose influx and blood flow in retina and brain of diabetic rats. J Cereb Blood Flow Metab. 2004;24:449–57.CrossRefPubMedGoogle Scholar
  35. 35.
    Nabi IR, Mathews AP, Cohen-Gould L, Gundersen D, Rodriguez-Boulan E. Immortalization of polarized rat retinal pigment epithelium. J Cell Sci. 1993;104:37–49.PubMedGoogle Scholar
  36. 36.
    Yamaoka K, Tanigawara Y, Nakagawa T, Uno T. A pharmacokinetic analysis program (MULTI) for microcomputer. Aust J Pharm. 1981;4:879–85.Google Scholar
  37. 37.
    Cook NJ, Molday LL, Reid D, Kaupp UB, Molday RS. The cGMP-gated channel of bovine rod photoreceptors is localized exclusively in the plasma membrane. J Biol Chem. 1989;264:6996–9.PubMedGoogle Scholar
  38. 38.
    Frasson M, Sahel JA, Fabre M, Simonutti M, Dreyfus H, Picaud S. Retinitis pigmentosa: rod photoreceptor rescue by a calcium-channel blocker in the rd mouse. Nat Med. 1999;5:1183–7.CrossRefPubMedGoogle Scholar
  39. 39.
    Takano Y, Ohguro H, Dezawa M, Ishikawa H, Yamazaki H, Ohguro I, et al. Study of drug effects of calcium channel blockers on retinal degeneration of rd mouse. Biochem Biophys Res Commun. 2004;313:1015–22.CrossRefPubMedGoogle Scholar
  40. 40.
    Bauer M, Karch R, Tournier N, Cisternino S, Wadsak W, Hacker M, et al. Assessment of P-glycoprotein transport activity at the human blood-retina barrier with (R)-11C-verapamil PET. J Nucl Med. 2017;58:678–81.CrossRefPubMedGoogle Scholar
  41. 41.
    Zhang Z, Uchida Y, Hirano S, Ando D, Kubo Y, Auriola S, et al. Inner blood-retinal barrier dominantly expresses breast cancer resistance protein: comparative quantitative targeted absolute proteomics study of CNS barriers in pig. Mol Pharm. 2017;14:3729–38.CrossRefPubMedGoogle Scholar
  42. 42.
    Fujii S, Setoguchi C, Kawazu K, Hosoya K. Functional characterization of carrier-mediated transport of pravastatin across the blood-retinal barrier in rats. Drug Metab Dispos. 2015;43:1956–9.CrossRefPubMedGoogle Scholar
  43. 43.
    Katayama K, Ohshima Y, Tomi M, Hosoya K. Application of microdialysis to evaluate the efflux transport of estradiol 17-beta glucuronide across the rat blood-retinal barrier. J Neurosci Methods. 2006;156:249–56.CrossRefPubMedGoogle Scholar
  44. 44.
    Hosoya K, Makihara A, Tsujikawa Y, Yoneyama D, Mori S, Terasaki T, et al. Roles of inner blood-retinal barrier organic anion transporter 3 in the vitreous/retina-to-blood efflux transport of p-aminohippuric acid, benzylpenicillin, and 6-mercaptopurine. J Pharmacol Exp Ther. 2009;329:87–93.CrossRefPubMedGoogle Scholar
  45. 45.
    Akanuma S, Hirose S, Tachikawa M, Hosoya K. Localization of organic anion transporting polypeptide (Oatp) 1a4 and Oatp1c1 at the rat blood-retinal barrier. Fluids Barriers CNS. 2013;10:29.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Han YH, Sweet DH, Hu DN, Pritchard JB. Characterization of a novel cationic drug transporter in human retinal pigment epithelial cells. J Pharmacol Exp Ther. 2001;296:450–7.PubMedGoogle Scholar
  47. 47.
    Lelong IH, Guzikowski AP, Haugland RP, Pastan I, Gottesman MM, Willingham MC. Fluorescent verapamil derivative for monitoring activity of the multidrug transporter. Mol Pharmacol. 1991;40:490–4.PubMedGoogle Scholar

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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