Molecular cloning and functional characterization of the OCTN2 transporter at the RBE4 cells, an in vitro model of the blood-brain barrier

Anne Friedrich, Puttur D. Prasad, Dorette Freyer, Vadivel Ganapathy, Peter Brust

Research output: Contribution to journalArticle

33 Scopus citations

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 μM 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.

Original languageEnglish (US)
Pages (from-to)69-79
Number of pages11
JournalBrain Research
Volume968
Issue number1
DOIs
StatePublished - Apr 4 2003

Keywords

  • Acetyl-L-carnitine
  • Blood-brain barrier
  • Carnitine transporter
  • Gene expression
  • In vitro model
  • MPP
  • Organic cation transporter
  • Propionyl-L-carnitine
  • TEA

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Clinical Neurology
  • Developmental Biology

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