MRNA expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines; a new in vitro BBB model for drug targeting

K. I. Hosoya, T. Takashima, K. Tetsuka, T. Nagura, S. Ohtsuki, H. Takanaga, M. Ueda, N. Yanai, M. Obinata, T. Terasaki

Research output: Contribution to journalArticlepeer-review

86 Citations (Scopus)

Abstract

Brain capillary endothelial cell lines (TR-BBB) were established from a recently developed transgenic rat harboring temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat) and used to characterize the endothelial marker, transport activity, and mRNA expression of transporters and tight-junction strand proteins at the blood-brain barrier (BBB). These cell lines expressed active large T-antigen and grew well at 33°C with a doubling-time of about 22-31 hr, but did not grow at 39°C. TR-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. Although the γ-glutamyltranspeptidase activity in TR-BBBs was ∼3% of that of the brain capillary fraction of a normal rat, it was localized in the apical side, suggesting that it reflects the functional polarity of the in vivo BBB. The mRNA of tight-junction strand proteins such as claudine-5, occludin, and junctional adhesion molecule are expressed in TR-BBB13. Drug efflux transporter, P-glycoprotein, with a molecular weight of 170 kDa was expressed in all TR-BBBs and mdr la, mdr lb, and mdr 2 mRNA were detected in TR-BBBs using RT-PCR. Moreover, mrp1 mRNA was expressed in all TR-BBBs. Influx transporter, GLUT-1, expressed at 55 kDa was revealed by Western blot analysis. It had 3-O-methyl-D-glucose (3-OMG) uptake activity which was concentration-dependent with a Michaelis-Menten constant of 9.86 ±1.20 mM. The mRNA of large neutral amino acid transporter, which consists of LAT-1 and 4F2hc was expressed in TR-BBBs. In conclusion, the conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB) had endothelial makers, expressed mRNA for tight-junction strand proteins and the influx and efflux transporters and produced GLUT-1, which is capable of 3-OMG transport activity.

Original languageEnglish
Pages (from-to)357-370
Number of pages14
JournalJournal of Drug Targeting
Volume8
Issue number6
DOIs
Publication statusPublished - 2000 Jan 1

Keywords

  • Blood-brain barrier
  • GLUT-1
  • P-glycoprotein
  • Temperature-sensitive SV 40T antigen
  • Tight-junction strand protein

ASJC Scopus subject areas

  • Pharmaceutical Science

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