Molecular and Physiological Evidence for Multifunctionality of Carnitine / Organic Cation Transporter OCTN 2

OCTN2 is an Na-dependent transporter for carnitine, which is essential for fatty acid metabolism, and its functional defect leads to fatal systemic carnitine deficiency (SCD). It also transports the organic cation tetraethylammonium (TEA) in an Naindependent manner. Here, we studied the multifunctionality of OCTN2, by examining the transport characteristics in cells transfected with mouse OCTN2 and in juvenile visceral steatosis (jvs) mice that exhibit a SCD phenotype owing to mutation of the OCTN2 gene. The physiological significance of OCTN2 as an organic cation transporter was confirmed by using jvs mice. The embryonic fibroblasts from jvs mice exhibited significantly decreased transport of [C]TEA. Pharmacokinetic analysis of [C]TEA disposition demonstrated that jvs mice showed decreased tissue distribution and renal secretory clearance. In transport experiments using OCTN2-expressing cells, TEA and carnitine showed mutual trans-stimulation effects in their transport, implying a carnitine/TEA exchange mechanism. In addition, Na affected the affinity of carnitine for OCTN2, whereas Na is unlikely to be involved in TEA transport. This is the first molecular and physiological demonstration of the operation of an organic cation transporter in renal apical membrane. The results are consistent with the physiological coupling of carnitine reabsorption with the secretion of organic cations. Organic cations such as tetraethylammonium (TEA) are distributed into various tissues and excreted into urine by specific transport systems in kidney. Previous studies suggested that organic cations are excreted by potential-dependent OCT transporters and by an organic cation/H antiporter across the renal epithelial basolateral and apical membranes, respectively (Zhang et al., 1998; Koepsell et al., 1999; Sweet and Pritchard, 1999), whereas no apically expressed transporters have been molecularly identified. We have isolated and characterized the new family of organic cation transporters, OCTN. The first member of the OCTN family, OCTN1, cloned from human fetal liver, transports organic cations such as TEA in a pH-dependent manner (Tamai et al., 1997; Yabuuchi et al., 1999). The second member, OCTN2, transports physiologically important carnitine in an Na-dependent manner, as well as organic cations in an Na-independent manner (Tamai et al., 1998; Wu et al., 1998, 1999; Ohashi et al., 1999). OCTN2 is present in various tissues, including kidney, skeletal muscle, heart, placenta, and others (Tamai et al., 1998). In 1988, the homozygous mutant mice, named juvenile visceral steatosis (jvs) mice, which exhibit cardiac hypertrophy, lipid accumulation in the liver, and hyperammonemia, (Koizumi et al., 1988; Horiuchi et al., 1993) were found with several histological changes (Koizumi et al., 1988; Narama et al., 1997) and alteration of carnitine disposition (Yokogawa et al., 1999a). The significance of OCTN2 as the carnitine transporter was clearly demonstrated using jvs mice, which exhibit the phenotype of systemic carnitine deficiency (SCD) caused by mutation of the OCTN2 gene (Leu352Arg) (Hashimoto et al., 1998; Lu et al., 1998; Nezu et al., 1999; Yokogawa et al., 1999c). However, no precise studies on organic cation transport by mouse OCTN2 have been done, and the physiological and pharmacological relevance of OCTN2 as an organic cation transporter remains to be clarified. Furthermore, the mechanism of the multifunctionality of OCTN2, transporting carnitine and organic cations in Na-dependent and independent manners, respectively, is unclear. Because the jvs mice have genetically mutated OCTN2 and completely lack carnitine transport activity, they are expected to be useful to evaluate a contribution of OCTN2 as an organic cation transporter. This study was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan. ABBREVIATIONS: TEA, tetraethylammonium; jvs, juvenile visceral steatosis; SCD, systemic carnitine deficiency; MPP, 1-methyl-4-phenylpyridinium; HEK, human embryonic kidney; NMG, N-methylglucamine; GFR, glomerular filtration rate; AUC, area under plasma concentration-time curve; CLr, renal clearance; CLs, renal secretory clearance; Kp, tissue-to-plasma concentration ratio. 0026-895X/01/5902-358–366$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 2 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 400/881215 Mol Pharmacol 59:358–366, 2001 Printed in U.S.A. 358 at A PE T Jornals on M ay 5, 2017 m oharm . aspeurnals.org D ow nladed from In the present study, we studied the multifunctionality of OCTN2 and the significance of OCTN2-mediated tissue distribution and renal excretion of the organic cation TEA by comparing the dispositions in wild type and jvs mice, as well as by examining transport in OCTN2-expressing cells. Experimental Procedures Materials. [Methyl-H]acetyl-L-carnitine hydrochloride (65 Ci/ mmol) and L-[methyl-H]carnitine hydrochloride (85 Ci/mmol) were purchased from Moravek Biochemicals Inc. (Brea, CA). [1-C]Tetraethylammonium bromide (2.4 mCi/mmol), [N-methyl-H]1-methyl4-phenylpyridinium acetate (MPP) (82 Ci/mmol), [N-methylH]verapamil hydrochloride (78.6 Ci/mmol) and [glycyl-2-H]paminohippurate (2.45 Ci/mmol) were from New England Nuclear (Boston, MA). [Pyridinyl-5-H]pyrilamine (28 Ci/mmol) was from Amersham Pharmacia Biotech (Buckinghamshire, UK). Other reagents, including cefazolin sodium, were obtained from Sigma Chemical Co. (St. Louis, MO), Wako Pure Chemical Industries (Osaka, Japan), and Nacalai Tesque, Inc. (Kyoto, Japan) and used without further purification. HEK 293 cells were obtained from Japanese Cancer Research Resources Bank (Tokyo, Japan). Studies of Uptake and Efflux by Fibroblasts and HEK 293 Cells Expressing Mouse OCTN2. Primary cultured fibroblasts from homozygous jvs and normal C57BL/6J embryos were prepared and cultured as described previously (Hashimoto et al., 1998). Briefly, the cells obtained were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum (Life Technologies, Tokyo, Japan) on plastic disks in humidified 5% CO2 at 37°C and used for uptake experiments. In the transport measurement, the cells cultured on plastic disks were incubated with transport medium (Hanks’ balanced salt solution) containing a radiolabeled test compound to initiate the uptake study. After an appropriate time, the disks were washed three times with ice-cold Hanks’ balanced salt solution, solubilized with 1 N NaOH, and the radioactivity was measured in a liquid scintillation counter after neutralization with HCl and addition of liquid scintillation fluid, Clear-sol I (Nacalai