Pharmaceutical Research

, Volume 24, Issue 4, pp 688–694 | Cite as

Establishing a Method to Isolate Rat Brain Capillary Endothelial Cells by Magnetic Cell Sorting and Dominant mRNA Expression of Multidrug Resistance-associated Protein 1 and 4 in Highly Purified Rat Brain Capillary Endothelial Cells

  • Sumio Ohtsuki
  • Hirofumi Yamaguchi
  • Tomoko Asashima
  • Tetsuya Terasaki
Research Paper

Purpose

To establish a method for isolating highly purified brain capillary endothelial cells (BCECs) from rat brain by using magnetic cell sorting, and clarify the expression levels of multidrug resistance-associated protein (Mrp) subtypes in these highly purified BCECs.

Methods

The cells were prepared from the capillary enriched-fraction by enzyme digestion, and reacted with anti-PECAM-1 antibody. The cell sorting was performed by autoMACS. The mRNA levels were measured by quantitative real-time PCR analysis.

Results

From five rats, 2.3 × 106 cells were isolated in the PECAM-1(+) fraction and the percentage of labeled cells in this was 85.9%. PECAM-1, claudin-5 and Tie-2 mRNA were concentrated in the PECAM-1(+) fraction compared with rat brain. The contamination by neurons and astrocytes was markedly less than in the brain capillary fraction prepared by the glass bead column method. Mrp1 and 4 were predominantly expressed in the PECAM-1(+) fraction at similar levels to Mdr1a. The mRNA levels of Mrp5 and 3 were 10.6 and 7.60% of that of Mrp1, respectively.

Conclusions

This new purification method provides BCECs with less contamination by neural cells. In the isolated BCECs, Mrp1 and 4 are predominantly expressed, suggesting that they play an important role at the rat blood-brain barrier.

Key words

brain capillary endothelial cells magnetic cell sorting multidrug resistance-associated protein PECAM-1 purification 

Abbreviations

6-mp

6-mercaptopurine

ABC

ATP binding cassette

AZT

azidothymidine

BBB

blood-brain barrier

BCECs

brain capillary endothelial cells

DHEAS

dehydroepiandrosterone sulfate

E217βG

17β-estradiol-D-17β-glucuronide

FL2

fluorescence channel 2

Mrp

multidrug resistance-associated protein

PBS

phosphate buffered saline

PE

Phycoerythrin

PECAM-1

platelet endothelial cellular adhesion molecule-1

SSC

side scattered light

References

  1. 1.
    G. D. Kruh and M. G. Belinsky. The MRP family of drug efflux pumps. Oncogene 22:7537–7552 (2003).PubMedCrossRefGoogle Scholar
  2. 2.
    Y. Zhang, H. Han, W. F. Elmquist, and D. W. Miller. Expression of various multidrug resistance-associated protein (MRP) homologues in brain microvessel endothelial cells. Brain Res. 876:148–153 (2000).PubMedCrossRefGoogle Scholar
  3. 3.
    M. Leggas, M. Adachi, G. L. Scheffer, D. Sun, P. Wielinga, G. Du, K. E. Mercer, Y. Zhuang, J. C. Panetta, B. Johnston, R. J. Scheper, C. F. Stewart, and J. D. Schuetz. Mrp4 confers resistance to topotecan and protects the brain from chemotherapy. Mol. Cell. Biol. 24:7612–7621 (2004).PubMedCrossRefGoogle Scholar
  4. 4.
    H. Potschka, M. Fedrowitz, and W. Loscher. Multidrug resistance protein MRP2 contributes to blood-brain barrier function and restricts antiepileptic drug activity. J. Pharmacol. Exp. Ther. 306:124–131 (2003).PubMedCrossRefGoogle Scholar
  5. 5.
    D. Sugiyama, H. Kusuhara, Y. J. Lee, and Y. Sugiyama. Involvement of multidrug resistance associated protein 1 (Mrp1) in the efflux transport of 17β estradiol-D-17β-glucuronide (E217βG) across the blood-brain barrier. Pharm. Res. 20:1394–1400 (2003).PubMedCrossRefGoogle Scholar
  6. 6.
    K. Hosoya, S. Ohtsuki, and T. Terasaki. Recent advances in the brain-to-blood efflux transport across the blood-brain barrier. Int. J. Pharm. 248:15–29 (2002).PubMedCrossRefGoogle Scholar
  7. 7.
    C. Chen, A. L. Slitt, M. Z. Dieter, Y. Tanaka, G. L. Scheffer, and C. D. Klaassen. Up-regulation of Mrp4 expression in kidney of Mrp2-deficient TR-rats. Biochem. Pharmacol. 70:1088–1095 (2005).PubMedCrossRefGoogle Scholar
  8. 8.
    B. M. Johnson, P. Zhang, J. D. Schuetz, and K. L. Brouwer. Characterization of transport protein expression in multidrug resistance-associated protein (mrp) 2-deficient rats. Drug Metab. Dispos. 34:556–562 (2005).PubMedCrossRefGoogle Scholar
  9. 9.
    H. Gutmann, M. Torok, G. Fricker, J. Huwyler, C. Beglinger, and J. Drewe. Modulation of multidrug resistance protein expression in porcine brain capillary endothelial cells in vitro. Drug Metab. Dispos. 27:937–941 (1999).PubMedGoogle Scholar
  10. 10.
    S. Seetharaman, M. A. Barrand, L. Maskell, and R. J. Scheper. Multidrug resistance-related transport proteins in isolated human brain microvessels and in cells cultured from these isolates. J. Neurochem. 70:1151–1159 (1998).PubMedCrossRefGoogle Scholar
  11. 11.
    Y. Zhang, J. D. Schuetz, W. F. Elmquist, and D. W. Miller. Plasma membrane localization of multidrug resistance-associated protein homologs in brain capillary endothelial cells. J. Pharmacol. Exp. Ther. 311:449–455 (2004).PubMedCrossRefGoogle Scholar
  12. 12.
    J. Hirrlinger, J. Konig, and R. Dringen. Expression of mRNAs of multidrug resistance proteins (Mrps) in cultured rat astrocytes, oligodendrocytes, microglial cells and neurones. J. Neurochem. 82:716–719 (2002).PubMedCrossRefGoogle Scholar
  13. 13.
    S. Mazzetti, L. Librizzi, S. Frigerio, M. de Curtis, and L. Vitellaro-Zuccarello. Molecular anatomy of the cerebral microvessels in the isolated guinea-pig brain. Brain Res. 999:81–90 (2004).PubMedCrossRefGoogle Scholar
  14. 14.
    R. J. Boado and M. M. Pardridge. A one-step procedure for isolation of poly(A)+ mRNA from isolated brain capillaries and endothelial cells in culture. J. Neurochem. 57:2136–2139 (1991).PubMedCrossRefGoogle Scholar
  15. 15.
    K. Morita, H. Sasaki, M. Furuse, and S. Tsukita. Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells. J. Cell Biol. 147:185–194 (1999).PubMedCrossRefGoogle Scholar
  16. 16.
    T. M. Schlaeger, S. Bartunkova, J. A. Lawitts, G. Teichmann, W. Risau, U. Deutsch, and T. N. Sato. Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice. Proc. Natl. Acad. Sci. U. S. A. 94:3058–3063 (1997).PubMedCrossRefGoogle Scholar
  17. 17.
    D. Scholz and J. Schaper. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is localized over the entire plasma membrane of endothelial cells. Cell Tissue Res. 290:623–631 (1997).PubMedCrossRefGoogle Scholar
  18. 18.
    M. Demeule, M. Labelle, A. Regina, F. Berthelet, and R. Beliveau. Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms. Biochem. Biophys. Res. Commun. 281:827–834 (2001).PubMedCrossRefGoogle Scholar
  19. 19.
    P. W. Hewett and J. C. Murray. Immunomagnetic purification of human microvessel endothelial cells using Dynabeads coated with monoclonal antibodies to PECAM-1. Eur. J. Cell Biol. 62:451–454 (1993).PubMedGoogle Scholar
  20. 20.
    M. Tomi and K. Hosoya. Application of magnetically isolated rat retinal vascular endothelial cells for the determination of transporter gene expression levels at the inner blood-retinal barrier. J. Neurochem. 91:1244–1248 (2004).PubMedCrossRefGoogle Scholar
  21. 21.
    Z. Wu, F. M. Hofman, and B. V. Zlokovic. A simple method for isolation and characterization of mouse brain microvascular endothelial cells. J. Neurosci. Methods 130:53–63 (2003).PubMedCrossRefGoogle Scholar
  22. 22.
    R. Bandopadhyay, C. Orte, J. G. Lawrenson, A. R. Reid, S. De Silva, and G. Allt. Contractile proteins in pericytes at the blood-brain and blood-retinal barriers. J. Neurocytol. 30:35–44 (2001).PubMedCrossRefGoogle Scholar
  23. 23.
    A. T. Nies, G. Jedlitschky, J. Konig, C. Herold-Mende, H. H. Steiner, H. P. Schmitt, and D. Keppler. Expression and immunolocalization of the multidrug resistance proteins, MRP1-MRP6 (ABCC1-ABCC6), in human brain. Neuroscience 129:349–360 (2004).PubMedCrossRefGoogle Scholar
  24. 24.
    H. C. Cooray, C. G. Blackmore, L. Maskell, and M. A. Barrand. Localisation of breast cancer resistance protein in microvessel endothelium of human brain. NeuroReport 13:2059–2063 (2002).PubMedCrossRefGoogle Scholar
  25. 25.
    S. Hori, S. Ohtsuki, M. Tachikawa, N. Kimura, T. Kondo, M. Watanabe, E. Nakashima, and T. Terasaki. Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte-derived soluble factor(s). J. Neurochem. 90:526–536 (2004).PubMedCrossRefGoogle Scholar
  26. 26.
    M. Tachikawa, M. Watanabe, S. Hori, M. Fukaya, S. Ohtsuki, T. Asashima, and T. Terasaki. Distinct spatio-temporal expression of ABCA and ABCG transporters in the developing and adult mouse brain. J. Neurochem. 95:294–304 (2005).PubMedCrossRefGoogle Scholar
  27. 27.
    D. Sugiyama, H. Kusuhara, Y. Shitara, T. Abe, P. J. Meier, T. Sekine, H. Endou, H. Suzuki, and Y. Sugiyama. Characterization of the efflux transport of 17β-estradiol-D-17β-glucuronide from the brain across the blood-brain barrier. J. Pharmacol. Exp. Ther. 298:316–322 (2001).PubMedGoogle Scholar
  28. 28.
    Z. S. Chen, K. Lee, and G. D. Kruh. Transport of cyclic nucleotides and estradiol 17-beta-D-glucuronide by multidrug resistance protein 4. Resistance to 6-mercaptopurine and 6-thioguanine. J. Biol. Chem. 276:33747–33754 (2001).PubMedCrossRefGoogle Scholar
  29. 29.
    M. Suzuki, H. Suzuki, Y. Sugimoto, and Y. Sugiyama. ABCG2 transports sulfated conjugates of steroids and xenobiotics. J. Biol. Chem. 278:22644–22649 (2003).PubMedCrossRefGoogle Scholar
  30. 30.
    H. Asaba, K. Hosoya, H. Takanaga, S. Ohtsuki, E. Tamura, T. Takizawa, and T. Terasaki. Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2. J. Neurochem. 75:1907–1916 (2000).PubMedCrossRefGoogle Scholar
  31. 31.
    Y. J. Lee, H. Kusuhara, J. W. Jonker, A. H. Schinkel, and Y. Sugiyama. Investigation of efflux transport of dehydroepiandrosterone sulfate and mitoxantrone at the mouse blood-brain barrier: a minor role of breast cancer resistance protein. J. Pharmacol. Exp. Ther. 312:44–52 (2005).PubMedCrossRefGoogle Scholar
  32. 32.
    N. Zelcer, G. Reid, P. Wielinga, A. Kuil, I. van der Heijden, J. D. Schuetz, and P. Borst. Steroid and bile acid conjugates are substrates of human multidrug-resistance protein (MRP) 4. Biochem. J. 371:361–367 (2003).PubMedCrossRefGoogle Scholar
  33. 33.
    Z. S. Chen, K. Lee, and G. D. Kruh. Transport of cyclic nucleotides and estradiol 17-β-D-glucuronide by multidrug resistance protein 4. Resistance to 6-mercaptopurine and 6-thioguanine. J. Biol. Chem. 276:33747–33754 (2001).PubMedCrossRefGoogle Scholar
  34. 34.
    J. D. Schuetz, M. C. Connelly, D. Sun, S. G. Paibir, P. M. Flynn, R. V. Srinivas, A. Kumar, and A. Fridland. MRP4: a previously unidentified factor in resistance to nucleoside-based antiviral drugs. Nat. Med. 5:1048–1051 (1999).PubMedCrossRefGoogle Scholar
  35. 35.
    Y. Deguchi, Y. Yokoyama, T. Sakamoto, H. Hayashi, T. Naito, S. Yamada, and R. Kimura. Brain distribution of 6-mercaptopurine is regulated by the efflux transport system in the blood-brain barrier. Life Sci. 66:649–662 (2000).PubMedCrossRefGoogle Scholar
  36. 36.
    K. Takasawa, T. Terasaki, H. Suzuki, and Y. Sugiyama. In vivo evidence for carrier-mediated efflux transport of 3′-azido-3′-deoxythymidine and 2′,3′-dideoxyinosine across the blood-brain barrier via a probenecid-sensitive transport system. J. Pharmacol. Exp. Ther. 281:369–375 (1997).PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Sumio Ohtsuki
    • 1
    • 2
    • 3
  • Hirofumi Yamaguchi
    • 1
  • Tomoko Asashima
    • 1
    • 2
    • 3
  • Tetsuya Terasaki
    • 1
    • 2
    • 3
  1. 1.Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan
  2. 2.New Industry Creation Hatchery CenterTohoku UniversitySendaiJapan
  3. 3.CREST and SORST of the Japan Science and Technology Agency (JST)SaitamaJapan

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