Elsevier

Brain Research

Volume 968, Issue 1, 4 April 2003, Pages 69-79
Brain Research

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

https://doi.org/10.1016/S0006-8993(02)04271-3Get rights and content

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.

Introduction

l-Carnitine (4-N-trimethylamino-3-hydroxybutyric acid) is a small, water soluble molecule which serves as an essential cofactor in the translocation of long-chain fatty acids into mitochondria where they are subjected to β-oxidation [4]. Despite its obligatory function in energy production from long-chain fatty acids, carnitine is produced endogenously only in a limited number of tissues. Therefore, specific transport mechanisms are necessary to provide the cells with carnitine from the blood. The synthesis of carnitine from trimethyllysine includes four enzymatic reactions [31]. Besides in the liver and in the kidney, carnitine is also synthesized in the brain [16], [37] where it is suggested to fulfil a different role than in peripheral tissues [27]. The level of β-oxidation in the normal adult brain is very low [46] and the carnitine/acylcarnitine translocase from rat brain mitochondria has a higher specificity towards short and medium chain acyl derivatives than towards long-chain acyl derivatives [17]. It is postulated that the major function of this transporter in the adult brain is the translocation of acetyl moieties from mitochondria into the cytoplasm [27]. It has been demonstrated that acetyl-l-carnitine is utilized for the synthesis of acetylcholine [10], [48], [49]. Furthermore, acetyl-l-carnitine may modulate cerebral glucose utilization [1]. These functions may contribute to the beneficial effects of carnitine and its acyl esters in patients with neurodegenerative diseases [31], [38]. The neuroprotective properties of acetyl-l-carnitine have also been demonstrated in vitro on hippocampal cultures chronically treated with the β-amyloid fragment 25–35 [11].

However, the expression of γ-butyrobetaine hydroxylase (EC 1.14.11.1), the rate-limiting enzyme in the biosynthetic pathway of l-carnitine, in the brain is very low as shown by Northern blot analysis [42]. This suggests that the brain may depend on plasma carnitine supply. Neural cells accumulate l-carnitine by a saturable, stereoselective, and sodium-dependent transport process [14], [28], [54]. Recently a high-affinity carnitine transporter (OCTN2) has been cloned and functionally characterized [34], [40], [50]. Interestingly, OCTN2 belongs to the gene family of organic cation transporters and accordingly this transporter mediates the uptake of l-carnitine in a Na+-coupled manner and several organic cations in a Na+-independent manner [51]. The l-carnitine transport mediated by OCTN2 and CT1 is described to be decreased by acetyl-l-carnitine, γ-butyrobetaine and betaine [34], [40], [45]. In mice a third member of the OCTN family was found, so-called OCTN3, which is able to transport carnitine in a sodium-independent manner [41].

Different to other tissues, the brain nutrient supply does not depend solely on the cellular expression of specific transporters but also on the presence of the blood–brain barrier (BBB) and its specific properties. The tight junctions of the brain endothelium prevent the paracellular passage of small polar molecules such as carnitine from blood to brain [19]. The expression of specific transporters at the brain endothelium is therefore important for the brain nutrient supply. The transport of carnitine has been studied on immortalized rat brain endothelial (RBE4) cells and on primary cultures of endothelial capillary cells derived from porcine brain. The carnitine transport at RBE4 cells was found to be sodium-independent [23] and mainly located at the apical membrane [25]. At the basolateral side of the RBE4 cell cultures a decreasing effect of neutral amino acids and 2-amino-bicyclo(2,2,1)-heptane-2-carboxylic acid (BCH) on the carnitine uptake was observed [25]. Also in porcine brain endothelial cells, evidence for an asymmetrical uptake was found [24]. It was suggested that a novel sodium-independent transport system is present in the apical membrane whereas the transport across the basolateral membrane, which is diminished by sodium transport inhibitors and amino acids, may be mediated by amino acid transport system(s). The involvement of the sodium-dependent carnitine transporter OCTN2 was excluded in these studies since there was a lack of any effect of short- or medium-chain acylcarnitines as well as a missing effect of betaine on the carnitine uptake in the brain capillary endothelial cells [25]. In contrast, Kido et al. [18] have recently shown that l-carnitine as well as acetyl-l-carnitine is transported at the BBB in vitro as well as in vivo in a sodium-dependent manner. These authors also have shown the expression of OCTN2 in rat and human primary brain endothelial cells by an RT-PCR method. In the present study, we have characterized a sodium-dependent transporter of carnitine at RBE4 cells with functional and molecular approaches. Based on these data, we conclude that the OCTN2 transporter is expressed at RBE4 cells as an in vitro model of the BBB.

Section snippets

Materials

[Ethyl-1-14C]tetraethylammonium ([14C]TEA) bromide (specific radioactivity, 55 mCi/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO, USA). l-[3H]-Carnitine (specific radioactivity, 65 mCi/mmol), acetyl-l-[3H]-carnitine (specific radioactivity, 65 mCi/mmol) and propionyl-l-[3H]-carnitine (specific radioactivity, 65 mCi/mmol) were purchased from Moravek Biochemical (Brea, CA, USA). The purity of the radiochemicals was between 96% and 99% as supplied by the companies and the

Transport of l-[3H]-carnitine

The transport of l-[3H]-carnitine was studied at RBE4 cells which were grown on 24-well plates until they reached confluence. Fig. 1 shows the time course of the l-[3H]-carnitine uptake at a concentration of 20 nM. The uptake was measured at 37 °C and pH 7.4 in buffer with sodium and in buffer with isoosmotical replacement of sodium ions by NMDG. Replacement of sodium ions caused a significant inhibition of the uptake. The inhibition was about 68% at 4 h uptake measurement. The difference

Discussion

Carnitine has long been known for its stimulation of fatty acid oxidation which has been attributed to its function as a shuttle for activated fatty acids at the mitochondrial membrane [4]. It has recently been shown that a specific high-affinity transporter (OCTN2) is responsible for this function [34], [40], [50]. Carnitine has also been successfully used as a pharmacological agent for the treatment of chronic degenerative diseases of the brain [38]. However, very little is known about how

Acknowledgements

This work was supported in part by the National Institutes of Health grant HL64196 and the DFG (Br 1360/7-1). We thank Dr. Wei Huang and Dr. Ramesh Kekuda for helpful discussion and support.

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