Characterization of uptake of folates by rat and human blood–brain barrier endothelial cells Joa ˜o R. Arau ´jo, Pedro Gonc ¸alves, and Fa ´tima Martel* Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, Porto, Portugal Abstract. 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 þ 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 þ -, 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 FRa nor of proton-coupled folate transporter (PCFT) in RBE4 cells, and mRNA expression of RFC and PCFT, but not of FRa, 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. V C 2010 International Union of Biochemistry and Molecular Biology, Inc. Volume 36, Number 3, May/June 2010, Pages 201–209  E-mail: fmartel@med.up.pt Keywords: folate, methyltetrahydrofolate, blood-brain barrier, uptake 1. Introduction The blood–brain barrier (BBB) is constituted by the brain microvessel endothelial cells, which possess specialized fea- tures such as tight intercellular junctions that limit paracellu- lar permeability under physiological conditions, and that express a unique pattern of receptors, transporters, and nonselective drug export pumps that protect the CNS from a very large variety of potentially harmful hydrophobic com- pounds [1,2]. Hence, the BBB is essential for maintenance of the homeostasis of the CNS. Although the importance of the BBB is well-recognized, there is a paucity of information concerning the mechanisms of transport across this barrier, especially when compared with other organs such as the kidney, liver, and intestine. The reason for this lack of knowl- edge derives mainly from inadequate techniques available to study transport across the BBB. Most of the transport studies have been performed using in vivo animal models, primary cultures of brain capillary endothelial cells, or cocul- tures of brain microvessel endothelial cells and astrocytes. However, all these models present serious disadvantages [1,2]. In this context, the recent establishment of immortal- ized brain endothelial cell culture systems seems very inter- esting. The RBE4 and the hCMEC/D3 cell lines are two immortalized cerebral microvascular endothelial cell lines that retain most of the morphological characteristics of rat and human BBB endothelial cells, respectively, [1,2]. Folates (vitamin B 9 ), the generic name given to a family of water-soluble vitamins, play a critical role in the develop- ment, function, and repair of the CNS. These one-carbon donors are required in key biosynthetic processes in mam- malian cells (including those in the CNS): 1) the de novo synthesis of purine and thymidylate precursors of nucleic acids, being thus essential for the de novo production of RNA and DNA; 2) the initiation of protein synthesis in mito- chondria, and 3) the biosynthesis of methionine, the precur- sor of S-adenosylmethionine, which is required for methyla- tion of DNA, histones, and neurotransmitters [3–6]. The importance of folates in CNS homeostasis is supported by several observations. First, the deficiency of folates is impli- cated in a number or neurological disorders including Alzheimer’s disease [7,8], Parkinson’s disease [9], and cere- brovascular dysfunction [10,11], although some conflicting clinical evidence has been found for some of these associa- tions, namely, cerebrovascular disease and dementia-type disorders [12,13]. Also, cerebral folate deficiency has been *Address for correspondence: Fa ´tima Martel, Ph.D., Department of Biochemistry, Faculty of Medicine of Porto, 4200-319 Porto, Portugal. Tel.: þ351 22 5513624; Fax: þ351 22 5513624; E-mail: fmartel@med.up.pt. Received 10 November 2009; accepted 1 February 2010 DOI: 10.1002/biof.82 Published online 15 March 2010 in Wiley InterScience (www.interscience.wiley.com) 201