title: Molecular cloning and functional characterization of the OCTN2 transporter at the RBE4 cells, an in vitro model of the blood-brain barrier.
abstract: The transport of L-carnitine (4-N-trimethylamino-3-hydroxybutyric acid), a compound known to be transported by the organic cation transporter/carnitine transporter OCTN2, was studied in immortalized rat brain endothelial cells (RBE4). The cells were found to take up L-carnitine by a sodium-dependent process. This uptake process was saturable with an apparent Michaelis-Menten constant for L-carnitine of 54+/-10 microM and a maximal velocity of 215+/-35 pmol/mg protein/h. Besides L-carnitine, the cells also took up acetyl-L-carnitine and propionyl-L-carnitine in a sodium-dependent manner and TEA in a sodium-independent manner. RT-PCR with primers specific for the rat OCTN2 transporter revealed the existence of OCTN2 mRNA in RBE4 cells. Screening of a cDNA library from RBE4 cells with rat OCTN2 cDNA as a probe identified a positive clone which showed, when expressed in HeLa cells, the functional characteristics of OCTN2. The HeLa cells expressing the RBE4 OCTN2 cDNA showed a sixfold increase in L-carnitine uptake and a fourfold increase in TEA uptake in a sodium-containing buffer. Typical inhibitors for organic cation transporters (e.g. MPP(+) or TEA) showed an inhibitory effect on the transport of L-carnitine and TEA into the transfected cells. Similarly, unlabeled L-carnitine inhibited the transport of [3H]-L-carnitine and [14C]TEA in transfected HeLa cells. It is concluded that RBE4 cells, a widely used in vitro model of the blood-brain barrier (BBB), express the organic cation/carnitine transporter OCTN2.
authors.affiliation: Forschungszentrum Rossendorf, Dresden, Germany. friedric@mpi-cbg.de
authors.forename: Anne
authors.initials: A
authors.lastname: Friedrich
authors.forename: Puttur D
authors.initials: PD
authors.lastname: Prasad
authors.forename: Dorette
authors.initials: D
authors.lastname: Freyer
authors.forename: Vadivel
authors.initials: V
authors.lastname: Ganapathy
authors.forename: Peter
authors.initials: P
authors.lastname: Brust
mesh_terms: D015655:1-Methyl-4-phenylpyridinium / Q000493:pharmacokinetics; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D001812:Blood-Brain Barrier / Q000502:physiology*; D002331:Carnitine / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D002794:Choline / Q000493:pharmacokinetics; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004730:Endothelium, Vascular; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology; D008954:Models, Biological; D027701:Organic Cation Transport Proteins; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000074058:Solute Carrier Family 22 Member 5; D019789:Tetraethylammonium / Q000493:pharmacokinetics; D013997:Time Factors; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014162:Transfection; D014316:Tritium / Q000493:pharmacokinetics
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D027701:Organic Cation Transport Proteins; D012333:RNA, Messenger; C492020:SLC22A5 protein, human; D000074058:Solute Carrier Family 22 Member 5; D014316:Tritium; D019789:Tetraethylammonium; D002794:Choline; D015655:1-Methyl-4-phenylpyridinium; D002331:Carnitine