EXPRESSION, LOCALIZATION, AND FUNCTION OF THE CARNITINE TRANSPORTER OCTN2 (SLC22A5) IN HUMAN PLACENTA Markus Grube, Henriette Meyer zu Schwabedissen, Katrin Draber, Damaris Pra ¨ ger, Klaus-Uwe Mo ¨ ritz, Knud Linnemann, Christoph Fusch, Gabriele Jedlitschky, and Heyo K. Kroemer Peter Holtz Research Center of Pharmacology and Experimental Therapeutics, Department of Pharmacology (M.G., H.M.z.S., K.D., D.P., K.-U.M., G.J., H.K.K.) and Department of Neonatology (K.L., C.F.), Ernst Moritz Arndt University, Greifswald, Germany Received July 23, 2004; accepted October 4, 2004 ABSTRACT: L-carnitine is assumed to play an important role in fetal develop- ment, and there is evidence that carnitine is transported across the placenta. The protein involved in this transfer, however, has not been identified on a molecular level. We therefore characterized localization and function of the carnitine transporter OCTN2 in human placenta. Significant expression of OCTN2 mRNA was de- tected in human placenta applying real-time polymerase chain reaction technology. Confocal immunofluorescence microscopy using an antibody directed against the carboxy terminus of OCTN2 protein revealed that it is predominantly expressed in the apical membrane of syncytiotrophoblasts. This was confirmed by the costaining of organic anion-transporting polypeptide B and MRP2, which are known to be expressed mainly in the basal and apical syncytiotrophoblasts membrane, respectively. To further support this finding, we performed transport studies using basal and apical placenta membrane vesicles. We could demonstrate that the car- nitine uptake into the apical vesicles was about eight times higher compared with the basal ones. Moreover, this uptake was sodium- and pH-dependent with an apparent K m value of 21 M and inhib- ited by verapamil, which is in line with published data for recom- binant OCTN2. Finally, experiments using trophoblasts in cell cul- ture revealed that expression of OCTN2 paralleled human choriogonadotropin production and thus is modulated by cellular differentiation. In summary, we show expression and function of OCTN2 in human placenta. Moreover, several lines of evidence indicate that OCTN2 is localized in the apical membrane of syncy- tiotrophoblasts, thereby suggesting a major role in the uptake of carnitine during fetal development. Carnitine plays an important physiological role, in particular, in -oxidation because it facilitates long-chain fatty acid transport across the inner mitochondrial membrane. Moreover, carnitine is involved in intracellular coenzyme A homeostasis and functions as an antioxidant (Bremer, 1983; Arduini et al., 1992; Pons and De Vivo, 1995). Only a few organs like brain, liver, and kidney have the ability to biosyn- thesize carnitine (Bremer, 1983), whereas other tissues like skeletal and heart muscles, where -oxidation plays a major role in energy metabolism, are highly dependent on active carnitine uptake from blood to maintain their carnitine steady-state concentration (Siliprandi et al., 1989). Recent studies describe the organic cation transporter novel type II (OCTN2) as a high affinity uptake system for carnitine. The OCTN2 cDNA codes for 557 amino acids consisting of 12 putative transmem- brane domains with a predicted molecular mass of 63 kDa (Wu et al., 1998). The transport of carnitine is sodium-dependent (Tamai et al., 1998), whereas other compounds such as tetraethylammonium are transported by OCTN2 in a sodium-independent way (Ohashi et al., 2001). Besides its physiological function, OCTN2 is of pharmacological relevance. Drugs like verapamil, pyrilamine, and -lactam antibiotics have been characterized as substrates of OCTN2 and/or inhibitors of carnitine transport (Ohashi et al., 1999, 2001; Wu et al., 1999; Ganapathy et al., 2000). Using screening approaches, the OCTN2 mRNA was detected in kidney, heart, skeletal muscle, and placenta (Tamai et al., 1998). Although OCTN2 expression in the kidney seems to be mainly involved in carnitine reabsorption, expression in muscle cells is assumed to be responsible for carnitine homeostasis in this tissue. The physiological role is further supported by studies of systemic carnitine deficiency, which was associated with mutations in the OCTN2 gene (Vaz et al., 1999; Lamhonwah et al., 2002). Interestingly, previous studies indicated a limited capacity of the fetal organism for fatty acid oxidation, which is a consequence of incompletely matured carnitine biosynthesis (Novak et al., 1981). This assumption leads to the conclusion that placental uptake of carnitine is pivotal for the fetal organism (Schmidt-Sommerfeld et al., 1981). In fact, sodium-dependent carnitine uptake has been demonstrated for the human choriocarcinoma-derived JAR cell line and brush-border syncytiotrophoblast vesicles. The protein involved in placental uptake This work was supported by the German Federal Ministry of Education and Research (NBL3 program, reference 01 ZZ 0103) and by the Karl and Lore Klein-Stiftung, Oy-Mittelberg, Germany. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.104.001560. ABBREVIATIONS: FCS, fetal calf serum; DMEM, Dulbecco’s modified Eagle’s medium; PCR, polymerase chain reaction; OATP-B, organic anion-transporting polypeptide B; PBS, phosphate-buffered saline; hCG, human choriogonadotropin; TEA, tetraethylammonium. 0090-9556/05/3301-31–37$20.00 DRUG METABOLISM AND DISPOSITION Vol. 33, No. 1 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 1560/1189430 DMD 33:31–37, 2005 Printed in U.S.A. 31 at ASPET Journals on July 21, 2018 dmd.aspetjournals.org Downloaded from