Characterization of uptake of folates by rat and human blood-brain barrier endothelial cells.
This study aimed to characterize (3)H-folic acid ((3)H-FA) and (14)C-methyltetrahydrofolic acid ((14)C-MTHF) uptake by rat (RBE4) and human (hCMEC/D3) blood-brain barrier (BBB) endothelial cell lines. Uptake of (3)H-FA and (14)C-MTHF by RBE4 cells was time dependent and linear for the first 2 min of incubation; uptake by hCMEC/D3 cells showed a less marked time-dependency and a greater experimental variability. So, further experiments were performed in RBE4 cells only. Uptake of (3)H-FA was stimulated at acidic and alkaline pH, Na(+) dependent, stimulated when F(-) substituted for Cl(-), energy independent, inhibited by premetrexed, stimulated by cytochalasin D, and unaffected by MTHF, DIDS, SITS, methotrexate, monensin, and FA. Uptake of (14)C-MTHF was found to be pH-, Na(+)-, Cl(-)- and energy independent, inhibited by premetrexed and methotrexate, stimulated by cytochalasin D, and unaffected by FA, DIDS, SITS, monensin, and MTHF. RT-PCR analysis showed mRNA expression of reduced folate transporter (RFC), but neither of FRalpha nor of proton-coupled folate transporter (PCFT) in RBE4 cells, and mRNA expression of RFC and PCFT, but not of FRalpha, in hCMEC/D3 cells. In conclusion, both human and rat BBB endothelial cells show little capacity for (3)H-FA and (14)C-MTHF apical uptake. Hence, these cell lines do not appear to be a good model to study the transport of folates at the BBB.
Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, Porto, Portugal.
João R
JR
Araújo
Pedro
P
Gonçalves
Fátima
F
Martel
D000818:Animals; D001692:Biological Transport / Q000502:physiology*; D001812:Blood-Brain Barrier / Q000166:cytology*; D002250:Carbon Radioisotopes; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism*; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002250:Carbon Radioisotopes; C032418:5,11-methenyltetrahydrohomofolate; D005492:Folic Acid