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

Brain capillary endothelial cell lines (TR-BBB) were established from a rec ently developed transgenic rat harboring temperature-sensitive simian virus 40 (ts SV 50) large T-antigen gene (Tg rat) and used to characterize the e ndothelial 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 degreesC wi th a doubling-time of about 12-31 hr, but did not grow at 39 degreesC. TR-B BBs expressed the typical endothelial marker, von Willebrand factor, and ex hibited acetylated low-density lipoprotein uptake activity. Although the ga mma -glutamyltranspeptidase activity in TR-BBBs was similar to 13% of that of the brain capillary fraction of a normal rat, it was localized in the ap ical side, suggesting that it reflects the functional polarity of the in vi vo BBB. The mRNA of tight-junction strand proteins such as claudine-5, occl udin, and junctional adhesion molecule are expressed in TR-BBB13, Drug effl ux transporter, P-glycoprotein, with a molecular weight of 170 kDa was expr essed 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. Influ x transporter, GLUT-1, expressed at 55 kDa was revealed by Western blot ana lysis. It had 3-O-methyl-D-glucose (3-OMG) uptake activity which was concen tration-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 immortaliz ed rat brain capillary endothelial cell lines (TR-BBB) had endothelial make rs, expressed mRNA for tight-junction strand proteins and the influx acid e fflux transporters and produced GLUT-1, which is capable of 3-OMG transport activity.