Transport of choline and carnitine at the blood-brain barrier in vitro

Introduction:

Choline is the metabolic precursor of neurotransmitter acetylcholine and of the membrane phospholipid phosphatidylcholine. This amine is not synthesized by the brain and is therefore taken up from the blood. It is transported by a specific transport system which is much less characterized than the transporter for glucose and amino acids. Carnitine which is important for the intracellular transport of fatty acids has been found to inhibit the choline transport. On the other hand specific transporters for carnitine are known (OCTN1, OCTN2) which have not been described at the blood-brain-barrier (BBB) up to know. Therefore we have studied the transport of the two amines at an in vitro model of the BBB. In this study, we have demonstrated that at least two different transport systems for choline and carnitine are functionally expressed.
Methods:
The RBE4 cell line was used as an in-vitro model of the BBB and it has been shown that these cells express typical BBB markers such as gamma-glutamyltransferase and alkaline phosphatase(1). Tracer uptake studies were performed in 24 multi-well plates using buffers containing either 25 mM Tris/Hepes (pH 8.5 for choline uptake) or 25 mM Hepes/Tris (pH 7.5 for carnitine uptake), supplemented with 0.5% albumin, 5 mM glucose, 5.4 mM KCl, 1,8 mM CaCl₂, 0.8 mM MgSO₄ and 140 mM NaCl. Whenever Na⁺-free buffers were used, NMDG chloride replaced NaCl isoosmotically. Cells were washed before starting the uptake and then incubated at room temperature (choline uptake) or 37°C (carnitine uptake) with radiolabeled substrates added to the incubation buffer. K_m values and V_max values were calculated from the uptake data. Specific inhibitors of the transporters were added to the incubation buffer to study the specificity of the process.
An RBE4 cell cDNA library was constructed using the Superscript plasmid system. This plasmid cDNA library, grown on NitroPure transfer membranes, was screened by the colony screening method as described by Vogeli and Kaytes (2). The cDNA probe used for screening was 2.1 kb pair long and consisted of most of the coding region of rOCTN2 cDNA. The probe was labeled with [alpha-₃₂P]dCTP using the ready-to-go oligolabeling kit, and used to screen the RBE4 cDNA library under low stringency conditions. The cDNA was functionally expressed in HeLa cells by vaccinia virus expression system (3). Transport measurements were made at room temperature using the same uptake buffer as described above without albumin.
Results:
The RBE4 cells express a Na⁺-independent choline transporter. Hemicholinium, an inhibitor for the Na⁺-dependent choline transporter, also inhibits the choline uptake found in these cells while tetraethylammonium (TEA) does not. The uptake of choline is saturable with a K_m value of 22 µM and a V_max of 515 pmol/(mg protein * 10 min).
We also isolated a full-length OCTN2 clone from the RBE4 cDNA library. HeLa cells transfected with this clone express Na⁺-independent TEA uptake and Na⁺-dependent carnitine uptake. These transfected cells also take up fatty acid esters of carnitine like propionylcarnitine and acetylcarnitine. The K_m value for the carnitine uptake at 37°C in RBE4 cells is about 50 µM. Specific inhibitors for the OCTN2 transporter (MPP⁺ and TEA) inhibit the carnitine uptake in RBE4 cells.
Comments:
In summary, we have found two separate transporters for choline and carnitine are expressed in the RBE4 cells. While the choline transporter is expected to supply the brain with choline for neurotransmitter synthesis OCTN2 is described as an carnitine carrier which also transports short fatty acids as carnitine esters through the cell membrane. The expression of OCTN2 at the BBB may help to understand the way of supplying the brain with acetyl groups for the synthesis of acetylcholine ...