title: mRna expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines; a new in vitro BBB model for drug targeting.
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 degrees C with a doubling-time of about 22-31 hr, but did not grow at 39 degrees C. TR-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. Although the gamma-glutamyltranspeptidase activity in TR-BBBs was approximately 13% 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 1a, mdr 1b, 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.
authors.affiliation: Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
authors.forename: K I
authors.initials: KI
authors.lastname: Hosoya
authors.forename: T
authors.initials: T
authors.lastname: Takashima
authors.forename: K
authors.initials: K
authors.lastname: Tetsuka
authors.forename: T
authors.initials: T
authors.lastname: Nagura
authors.forename: S
authors.initials: S
authors.lastname: Ohtsuki
authors.forename: H
authors.initials: H
authors.lastname: Takanaga
authors.forename: M
authors.initials: M
authors.lastname: Ueda
authors.forename: N
authors.initials: N
authors.lastname: Yanai
authors.forename: M
authors.initials: M
authors.lastname: Obinata
authors.forename: T
authors.initials: T
authors.lastname: Terasaki
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D000957:Antigens, Viral, Tumor / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line / Q000166:cytology / Q000378:metabolism*; D062507:Claudin-5; D016503:Drug Delivery Systems / Q000379:methods*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D006151:Guanosine / Q000031:analogs & derivatives / Q000493:pharmacokinetics*; D062726:Junctional Adhesion Molecules; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D062793:Occludin; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000957:Antigens, Viral, Tumor; D015815:Cell Adhesion Molecules; D062507:Claudin-5; C491510:Cldn5 protein, rat; D051272:Glucose Transporter Type 1; D062726:Junctional Adhesion Molecules; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D062793:Occludin; C568987:Ocln protein, rat; D012333:RNA, Messenger; C495679:Slc2a1 protein, rat; C037626:3'-O-methylguanosine; D006151:Guanosine