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Research ArticleArticle

Na+-Dependent Carnitine Transport by Organic Cation Transporter (OCTN2): Its Pharmacological and Toxicological Relevance

Rikiya Ohashi, Ikumi Tamai, Hikaru Yabuuchi, Jun-Ichi Nezu, Asuka Oku, Yoshimichi Sai, Miyuki Shimane and Akira Tsuji
Journal of Pharmacology and Experimental Therapeutics November 1999, 291 (2) 778-784;

Abstract

Carnitine deficiency, either primary or drug-induced, causes critical symptoms and is thought to involve alteration of active transport of carnitine across the plasma membrane of tissues as the underlying mechanism. Recently, we showed that human organic cation transporter, hOCTN2, cloned as a member of the organic cation transporter family, is a physiologically important Na+-dependent high-affinity carnitine transporter in humans. In this study, we further characterized the functional properties of hOCTN2 and examined the interaction between hOCTN2-mediated carnitine transport and clinically used drugs to assess possible toxicological effects. When expressed in human embryonic kidney (HEK)293 cells, hOCTN2 showed low but significant stereospecific transport activity:d-carnitine was transported with lower affinity ( Km = 10.9 μM) than thel-isomer ( Km = 4.3 μM). One Na+ appeared to be associated with the transport of one carnitine molecule. hOCTN2-mediated transport of acetyl-l-carnitine was also Na+-dependent and of high affinity, with a Km value of 8.5 μM. To examine the transport activity for organic cations other than carnitine and the possible relationship of drug-induced carnitine deficiency with hOCTN2, the inhibitory effect of several drugs on hOCTN2-mediated l-carnitine transport was examined. Many zwitterionic drugs, such as cephaloridine, and many cationic drugs, such as quinidine and verapamil, exhibited significant inhibitory effects. Among these inhibitors, tetraethylammonium, pyrilamine, quinidine, verapamil, and valproate were found to be transported by hOCTN2. The results suggest that the carnitine deficiency-related toxicological effects by long-term treatment with such drugs might be ascribed to a functional alteration of hOCTN2-mediated carnitine transport.

l-Carnitine is a highly polar endogenous compound and plays a physiologically important role in the β-oxidation of fatty acids by facilitating the transport of long-chain fatty acids (Fritz and Yue, 1959; Bremer, 1983). Carnitine deficiency may cause cardiomyopathy, skeletal myopathy, hypoketotic coma, hypoglycemia, and hyperammonemia (Duran et al., 1990; Scholte et al., 1990; Pons and De Vivo, 1995). Primary carnitine deficiency is considered to be related to a defect of active transport of carnitine across the plasma membranes of tissues, whereas secondary carnitine deficiency seems to be associated with an impaired oxidation of acyl-coenzyme A intermediates in the mitochondria, a limited supply of substrates for carnitine biosynthesis, or a dietary insufficiency (Pons and De Vivo, 1995). In 1988, the juvenile visceral steatosis (jvs) mouse, which shows several symptoms of primary and/or secondary carnitine deficiency, was discovered, and was subsequently demonstrated to lack high-affinity carnitine transport activity in several tissues (Koizumi et al., 1988; Kuwajima et al., 1991, 1996;Horiuchi et al., 1993, 1994; Hashimoto et al., 1998). Carnitine deficiency can also occasionally be induced by long-term treatment with various drugs. The antiepileptic valproate induces hyperammonemia and Reye’s-like syndrome, both of which are serious side effects (Gerber et al., 1979; Ohtani et al., 1982). It has also been reported that the antibiotic pivampicillin induced hypoketotic hypoglycemia (Holme et al., 1989), and that the antiamebic emetine (Kuntzer et al., 1990) and the antivirotic zidovudine (Dalakas et al., 1994) induced myopathy. In the case of valproate, it was suggested that the induced carnitine deficiency is related to a transporter-mediated interaction between carnitine and the drug molecule (Tein et al., 1994, 1996). The causes of carnitine deficiency induced by emetine and zidovudine remain to be clarified. Accordingly, at least some cases of drug-induced carnitine deficiency may be due to drug interaction with the carnitine transporter.

Recently, we have isolated a Na+-dependent carnitine transporter, human organic cation transporter, hOCTN2, from human kidney (Tamai et al., 1998) as a homolog of the novel organic cation transporter hOCTN1 (Tamai et al., 1997). We have also found in jvs mice and carnitine-deficient patients that mutations of OCTN2 result in primary carnitine deficiency (Nezu et al., 1999). When expressed in human embryonic kidney (HEK)293 cells, hOCTN1 caused significant transport of tetraethylammonium (TEA), a typical organic cation, in a pH-dependent manner, which suggests that hOCTN1 may be an organic cation transporter whose activity is enhanced by an outwardly directed proton gradient or membrane potential difference at the renal epithelial apical membrane (Tamai et al., 1997). On the other hand, hOCTN2 exhibited a high activity of carnitine transport in a Na+-dependent manner, suggesting that hOCTN2 is a high-affinity, Na+/carnitine cotransporter. However, Wu et al. (1998) demonstrated that hOCTN2 transports TEA in a pH-dependent manner. hOCTN2 may transport organic cationic compounds as well as carnitine, which implies that carnitine transport via hOCTN2 may be inhibited by drugs that are recognized by hOCTN2.

Acetyl-l-carnitine, an endogenous homolog ofl-carnitine, is also physiologically important in supplying the acetyl moiety to the tricarboxylic acid cycle system to generate ATP and in transporting the acetyl moiety that is produced by the β-oxidation system to the mitochondrial outer membrane (Siliprandi et al., 1990). Interestingly, acetyl-l-carnitine has a protective effect against the neurotoxicities induced by ammonia (Matsuoka and Igisu, 1993), 1-methyl-4-phenylpyridinium (MPP) (Steffen et al., 1995) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Harik and Hritz, 1993). Acetyl-l-carnitine has been tested for the treatment of Alzheimer’s disease (Rai et al., 1990; Pettegrew et al., 1995) and Down’s syndrome (De Falco et al., 1994). Therefore, acetyl-l-carnitine is physiologically and pharmacologically important, with potential as a neuroprotective drug. Although the uptake of acetyl-l-carnitine by cultured human heart cells (CCL 27) was saturable and was inhibited by l-carnitine (Molstad et al., 1977), a transporter for acetyl-l-carnitine has not yet been identified. Thus, we wanted to clarify whether or not acetyl-l-carnitine is transported by hOCTN2.

The purposes of the present study were to examine the functional properties of hOCTN2, including substrate specificity, stereospecificity, and pH dependency, and to elucidate the pharmacological and toxicological relevance of hOCTN2 by studying the transport of various clinically used drugs by hOCTN2 and the effects of these drugs on hOCTN2-mediated carnitine transport.

Experimental Procedures

Materials.

[Methyl-3H]acetyl-l-carnitine hydrochloride (65 Ci/mmol), l-[methyl-3H]carnitine hydrochloride (85 Ci/mmol),d-[methyl-3H]carnitine hydrochloride (65 Ci/mmol), [14C]guanidine hydrochloride (55 mCi/mmol), [4,5-3H]valproate (45 Ci/mmol), and [3H(G)]actinomycin D (4.4 Ci/mmol) were purchased from Moravek Biochemicals Inc. (Brea, CA). [1-14C]TEA bromide (2.4 mCi/mmol), [N-methyl-3H]MPP (82 Ci/mmol), [N-methyl-3H]verapamil hydrochloride (78.6 Ci/mmol), and [glycyl-2-3H]p-aminohippurate (PAH) (2.45 Ci/mmol) were purchased from New England Nuclear (Boston, MA). [Pyridinyl-5-3H]pyrilamine (28 Ci/mmol) and [N-methyl-3H]cimetidine (11.3 Ci/mmol) were purchased from Amersham Int. (Buckinghamshire, England). [9-3H]Quinidine hydrochloride (15 Ci/mmol) was from American Radiolabeled Chemicals Inc. (St. Louis, MO). Other reagents were obtained from Sigma Chemical Co. (St. Louis, MO), Wako Pure Chemical Industries (Osaka, Japan), Nacalai Tesque, Inc. (Kyoto, Japan), or Funakoshi Co. (Tokyo, Japan). HEK293 cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan).

Transport Study in HEK293 Cells.

The full-length hOCTN2 cDNA was subcloned into the BamHI sites of the expression vector pcDNA3 (Invitrogen, San Diego, CA) and the construct, pcDNA3/hOCTN2, was used to transfect HEK293 cells by means of the calcium phosphate precipitation method as described previously (Tamai et al., 1997). The cells were cultivated in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (Gibco BRL, Toyko, Japan), 100 U/ml penicillin, and 100 μg/ml streptomycin in tissue culture dishes in a humidified incubator at 37°C under 5% CO2 for 24 h, and then transfected with pcDNA3 plasmid carrying full-length hOCTN2 cDNA or with the pcDNA3 plasmid vector alone. At 48 h after transfection, the cells were harvested by scraping with a rubber policemen and suspended in the transport medium, which consisted of 125 mM NaCl, 4.8 mM KCl, 5.6 mM d-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM HEPES (pH 7.4). The cell suspension and transport medium containing a radiolabeled test compound were preincubated separately for 20 min and then mixed to initiate uptake. At appropriate times, 200-μl aliquots of the mixture were withdrawn and the cells were separated from the transport medium by centrifugation in a microtube containing a silicon oil and liquid paraffin mixture with a density of 1.03 and 3 N KOH. The resultant cell pellets were solubilized in 3 N KOH and neutralized with HCl, and the associated radioactivity was quantitated in a liquid scintillation counter (Aloka, Tokyo, Japan). Cellular protein content was determined according to the method of Bradford (1976) using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) and BSA as the standard. When sodium ions were replaced with N-methylglucamine, the cells obtained were suspended in sodium-free medium. The composition of sodium-free medium was the same as that of the above transport medium except that the sodium chloride was replaced isotonically withN-methylglucamine chloride. When the transport was to be measured at an acidic or alkaline pH (pH 5.5–8.4), the pH was adjusted using HCl, NaOH, or KOH.

Data Analysis.

Initial uptake rates were usually obtained by measuring the uptake at 3 or 5 min and the uptake values were expressed as the cell-to-medium concentration (C/M) ratio (μl/mg protein/3 min or 5 min) obtained by dividing uptake amount by the concentration of test compound in the medium. To estimate kinetic parameters for saturable transport and the stoichiometry between Na+ andl-carnitine or acetyl-l-carnitine, the uptake rate was fitted to the following equations by means of nonlinear least-squares regression analysis using WinNonlin (Scientific Consulting Inc., Cary, NC).v=Vmax×s/(Km+s) Equation 1v′=Vmax×[Na+]n/{KmNa+n+[Na+]n} Equation 2where v and v′ and s are the uptake rates and concentration of substrate (acetyl-l-carnitine or d-carnitine), and Km, Vmax, and n are the half-saturation concentration (Michaelis constant), the maximum transport rate, and the Hill coefficient, respectively. All data were expressed as means ± S.E. and statistical analysis was performed by using Student’s t test. The criterion of significance was taken to be p < .05.

Results

Functional Characterization of Na+-Dependent Carnitine Transporter hOCTN2.

Because l-carnitine transport by hOCTN2 was inhibited by the d-isomer of carnitine with lower affinity than the l-isomer (Tamai et al., 1998), the transport of d-carnitine was examined to clarify the stereospecificity. The hOCTN2-mediated d-carnitine uptake by the HEK293 cells was saturable (Fig.1A). Eadie-Hofstee plots gave a single straight line (Fig. 1B), suggesting the participation of a single functional binding site for d-carnitine, as forl-carnitine (Tamai et al., 1998). The Km and Vmax values estimated by nonlinear least-squares regression analysis ford-carnitine transport according to Eq. 1 were 10.9 ± 0.609 μM and 3.17 ± 0.075 nmol/mg protein/3 min, respectively.

Figure 1
Figure 1

Concentration of d-carnitine uptake by hOCTN2-transfected HEK293 cells. A, uptake of d-carnitine was measured for 3 min at 37°C in transport buffer (pH 7.4). B, Eadie-Hofstee plot of the saturable component. Dotted and solid lines represent the apparently nonsaturable uptake obtained from cells transfected with the pcDNA3 plasmid vector alone (▵) and the saturable uptake obtained from nonlinear least-squares regression analysis of the hOCTN2-mediated uptake (●) after subtracting the apparently nonmediated uptake (▵) from the total uptake (○). Results are means ± S.E. of three determinations.

The pH dependence of hOCTN2-mediatedl-[3H]carnitine uptake in HEK293 cells is shown in Fig. 2A. When the pH in the transport medium was acidic, pH 5.5 or 6.0, hOCTN2-mediatedl-[3H]carnitine transport by HEK293 cells was significantly decreased to approximately 70 to 80% of that at neutral or alkaline pH (p < .05), whereas uptake by mock-transfected cells, which were transfected with pcDNA3-plasmid vector alone, was not affected by pH. Furthermore, the uptake at neutral pH was comparable with that at alkaline pH.

Figure 2
Figure 2

Extracellular pH (A) and Na+concentration (B) dependencies ofl-[3H]carnitine uptake by hOCTN2-transfected HEK293 cells. Uptake of l-[3H]carnitine (10 nM) by hOCTN2- (○) and pcDNA3 vector alone-transfected (▵) HEK293 cells was measured for 3 min at 37°C. Osmolality of the medium was adjusted by adding appropriate concentrations ofN-methylglucamine chloride. The sum of NaCl andN-methylglucamine chloride concentration in the medium was 125 mM. Results are means ± S.E. of three determinations.

Figure 2B shows the Na+-concentration dependence of hOCTN2-mediated l-[3H]carnitine uptake by HEK293 cells. The uptake rate ofl-[3H]carnitine increased with increasing concentration of Na+, and the carnitine uptake exhibited a simple hyperbolic curve as the Na+ concentration was increased. The estimated Hill coefficient (n) and KmNa+ according to Eq. 2 were 0.926 ± 0.411 and 18.5 ± 0.94 mM, respectively.

The inhibitory effects of acylcarnitines of various chain lengths on the hOCTN2-mediated l-[3H]carnitine uptake are shown in Fig. 3. The acylcarnitines with a long chain length, such as palmitoyl- and lauroyl-l-carnitine, inhibited l-carnitine uptake much more strongly than did acylcarnitines with a short chain length, such as acetyl- and propionyl-l-carnitine, i.e., increasing chain length of the acyl moiety was accompanied by an increasing inhibitory effect. Nonspecific membrane perturbation by these compounds could be eliminated, because l-carnitine uptake in cells transfected with expression vector alone was not increased by tested acylcarnitines.

Figure 3
Figure 3

Inhibitory effect of acylcarnitines with different chain lengths on hOCTN2. Uptake ofl-[3H]carnitine (10 nM) in the presence of various acylcarnitines at 5 and 50 μM was measured for 3 min at 37°C in transport buffer (pH 7.4). Results are means ± S.E. of three determinations; ∗, indicates a significant difference from the control studied in the absence of acylcarnitines. When the S.E. is not shown, it is smaller than the symbols.

hOCTN2-Mediated Transport Properties of Acetyl-l-carnitine.

The transport of acetyl-l-carnitine as a model compound of carnitine-like drugs was examined. Figure 4 shows the concentration dependence of hOCTN2-mediated acetyl-l-carnitine uptake. Eadie-Hofstee plots (Fig. 4B) gave a single straight line, as was found for l- andd-carnitine. The Km and Vmax values estimated for acetyl-l-carnitine transport were 8.50 ± 0.422 μM and 1.38 ± 0.026 nmol/mg protein/3 min, respectively. The pH dependence of hOCTN2-mediated [3H]acetyl-l-carnitine uptake in HEK293 cells is shown in Fig. 5A. When the pH in the transport medium was acidic (pH 5.5), [3H]acetyl-l-carnitine uptake was about 70% of that at pH 7.4. The Na+ concentration dependence of hOCTN2-mediated [3H]acetyl-l-carnitine uptake by HEK293 cells is shown in Fig. 5B. The uptake rate of [3H]acetyl-l-carnitine increased with an increasing concentration of Na+, and the acetyl-l-carnitine uptake exhibited a simple hyperbolic curve as the Na+ concentration was increased. The values of the Hill coefficient (n) and KmNa+were 0.989 ± 0.219 and 11.3 ± 2.89 mM, respectively.

Figure 4
Figure 4

Concentration dependence of acetyl-l-carnitine uptake by hOCTN2-transfected HEK293 cells. A, uptake of acetyl-l-carnitine was measured for 3 min at 37°C in transport buffer (pH 7.4). B, Eadie-Hofstee plot of the saturable component. Dotted and solid lines represent the apparently nonsaturable uptake obtained from cells transfected with the pcDNA3 plasmid vector alone (▵) and the saturable uptake obtained as described in the legend to Fig. 1. ○, total uptake. Results are means ± S.E. of three determinations.

Figure 5
Figure 5

Extracellular pH (A) and Na+concentration (B) dependencies of [3H]acetyl-l-carnitine uptake by hOCTN2-transfected HEK293 cells. Uptake of [3H]acetyl-l-carnitine (7.7 nM) by hOCTN2- (○) and pcDNA3 vector-transfected (▵) HEK293 cells was measured in the same way as described in the legend to Fig. 2. Results are means ± S.E. of three determinations.

Influence of Xenobiotics on l-Carnitine Transport by hOCTN2.

The effects of various xenobiotics on the hOCTN2-mediatedl-[3H]carnitine transport are summarized in Table 1. Various cationic compounds such as TEA, nicotine, MPP, pyrilamine, actinomycin D, cimetidine, clonidine, procainamide, quinidine, quinine, verapamil, and emetine showed significant inhibitory effects, whereas l-arginine, guanidine, and N′-methylnicotinamide (NMN) were not inhibitory. Furthermore, zwitterionic compounds such as ofloxacin, lomefloxacin, grepafloxacin, cephaloridine, ceftazidime, cefsulodin, trimethyllysine, and S-methylmethionine sulfonium significantly reduced carnitine uptake. Although most of the anionic compounds and dipeptide analogs tested were not inhibitory, probenecid and valproate were weakly inhibitory. Pivalate and zidovudine (AZT) were not inhibitory even at 500 μM.

View this table:
Table 1

Influence of several xenobiotics on carnitine transport by hOCTN2-transfected HEK293 cells

hOCTN2-Mediated Transport of Various Compounds.

Uptake of several cationic (TEA, MPP, guanidine, pyrilamine, cimetidine, quinidine, verapamil, and actinomycin D) and anionic (valproate and PAH) compounds by hOCTN2 was examined at 5 min. As shown in Table2, l- andd-carnitine and acetyl-l-carnitine were relatively good substrates for hOCTN2. Although native uptake by HEK293 cells observed in mock-transfected cells was rather high, quinidine and verapamil, which are potent inhibitors of hOCTN2-mediatedl-carnitine transport, showed significantly increased uptake by hOCTN2-transfected cells. Among moderate inhibitors, uptake of TEA and pyrilamine was increased in hOCTN2-transfected cells, whereas MPP, guanidine, cimetidine, and actinomycin D were not transported. The uptake of these cationic compounds did not increase on longer reaction (over 30 min) except in the case of TEA, for which the uptake was increased about 5-fold at 30 min (data not shown). Valproate, an anionic weak inhibitor of carnitine transport, showed a small but statistically significant increase of uptake in hOCTN2-transfected cells.

View this table:
Table 2

Xenobiotics uptake by hOCTN2-transfected HEK293 cells

The Na+ dependence of the uptake of the cationic compounds pyrilamine and verapamil by hOCTN2 was compared with those of carnitines (d- and l-carnitine and acetyl-l-carnitine; Fig. 6). In the presence of Na+ (125 mM NaCl), the carnitines and cationic compounds were all transported by hOCTN2. In the absence of Na+ (125 mM N-methylglucamine), uptakes of carnitines by hOCTN2 were significantly decreased compared with those in the presence of Na+, whereas the uptakes of pyrilamine and verapamil in the absence of Na+ were comparable with or higher than those in the presence of Na+.

Figure 6
Figure 6

Na+ dependence of transport of carnitines and organic cations by hOCTN2-transfected HEK293 cells. Uptakes ofl-[3H]carnitine (10 nM),d-[3H]carnitine (7.7 nM), [3H]aectyl-l-carnitine (7.7 nM), [3H]pyrilamine (17.9 nM), and [3H]verapamil (6.4 nM) were measured for 3 min at 37°C in transport buffer (pH 7.4). Results show values after subtracting the uptake by mock-transfected cells from that by the hOCTN2-transfected cells. Open and closed columns show the uptakes of carnitines and organic cations in the presence and absence of Na+ in the transport medium, respectively. Results are means ± S.E. of three determinations; ∗, indicates a significant difference from the presence of Na+ in the transport medium.

Discussion

We previously found that OCTN2, cloned as a homolog of the organic cation transporter OCTN1, transports carnitine in a high-affinity, Na+-dependent manner (Tamai et al., 1998). Furthermore, our recent findings demonstrated that mutations of OCTN2 are directly related to primary carnitine deficiency in the human as well as the mouse (Nezu et al., 1999). Accordingly, OCTN2 is a physiologically important transporter for carnitine metabolism, though its functional properties remain to be fully established. In the present study, we further characterized the carnitine transport properties of hOCTN2 and the influence of xenobiotics on hOCTN2-mediated carnitine transport.

To evaluate the hOCTN2-mediated carnitine transport properties, we studied the stereo specificity, pH dependence, and Na+concentration dependence of transport using hOCTN2-expressing HEK293 cells. d-Carnitine was transported in a Na+-dependent manner, like the l-isomer, but with a slightly lower affinity ( Km, 10.9 μM) compared with the l-isomer ( Km, 4.34 μM). Moderate pH dependence was observed in l-carnitine transport, with a maximum uptake activity between pH 7.0 and 8.2 and slightly but significantly lower transport at acidic pH. The stoichiometric analysis suggested that one Na+ is associated with the transport of one carnitine molecule, because the Hill coefficient (n) was 0.926. These results suggest that hOCTN2 exhibits moderate stereospecificity, slight pH dependence, and Na+ dependence with a one-to-one stoichiometry with carnitine. Wu et al. (1998) found that hOCTN2 is also an organic cation transporter, observing a pH-dependent TEA uptake. By using intact human placental choriocarcinoma cell line JAR cells derived from human placenta, Prasad et al. (1996) showed that carnitine transport is stereospecific, with a higher affinity for the l-isomer than the d-isomer, is associated with Na+-flux with a coupling ratio of 1:1, and has a KmNa+ of 6.8 mM. Furthermore Berardi et al. (1995) reported that rat renal Na+-dependent carnitine transport is pH dependent, with high activity at neutral and alkaline pH. At present, the underlying mechanisms for the pH effect of carnitine transport activity are not clear. Because the sodum ion-gradient has a predominant role as the driving force, we think that a carnitine/proton exchange mechanism is unlikely. Depolarization of the cells and/or the presence of optimal conformation of OCTN2 protein might explain the apparent pH-dependent change of carnitine transport activity. The results obtained in this study are in good agreement with these previous findings on carnitine transport in placenta and kidney, in which carnitine plays significant physiological roles.

Substrate specificity of hOCTN2 was assessed by examining the inhibitory effects of acylcarnitines of various chain lengths. Acylcarnitines with a longer chain length showed stronger inhibitory activity on hOCTN2-mediated carnitine transport (Fig. 3). Accordingly, greater lipophilicity of the acyl moiety may increase the affinity for hOCTN2. Similar acyl chain length dependence of the inhibitory effect on l-carnitine transport was reported by Molstad et al. (1977) using cultured human heart cells and by Berardi et al. (1995)using Xenopus laevis oocytes expressing total rat renal mRNA. Because acetyl-l-carnitine inhibited hOCTN2-mediated carnitine transport, we examined the transport of acetyl-l-carnitine by hOCTN2. Acetyl-l-carnitine transport was saturable with high affinity ( Km, 8.50 μM), which is close to that for l-carnitine transport ( Km, 4.34 μM). The Km of hOCTN2-mediated acetyl-l-carnitine uptake agreed well with the Km (8 μM) previously obtained in cultured human heart cells (Molstad et al., 1977). The pH profile and Hill coefficient (0.989) of hOCTN2-mediated acetyl-l-carnitine uptake were also similar with those of l-carnitine transport. These results suggest that acetyl-l-carnitine is transported by hOCTN2 in the same manner as l-carnitine, and hOCTN2 may influence the distribution of acetyl-l-carnitine in tissues. Because hOCTN2 is widely expressed in various tissues, with high expression in placenta, kidney, and heart (Tamai et al., 1998), it is likely that hOCTN2 plays a significant role in transporting carnitine and its acyl derivatives in these tissues.

From the viewpoint of the potential interaction between xenobiotics and carnitine on hOCTN2, we examined the effect of various xenobiotics on the l-carnitine transport by hOCTN2. The initial uptake ofl-[3H]carnitine was significantly inhibted by a number of xenobiotics (Table 1), especially lipophilic organic cations such as quinidine, quinine, verapamil, and emetine; zwitterionic compounds such as S-methylmethionine sulfonium; and the β-lactam antibiotics cephaloridine, ceftazidime, and cefsulodin. One of the mechanisms of nephrotoxicity caused by cephaloridine may be a reduction of renal mitochondrial acylcarnitine-mediated respiration, because cephaloridine reduced renal carnitine reabsorption and mitochondrial carnitine transport (Tune and Hsu, 1994). Therefore, the nephrotoxicity of cephaloridine may be at least partly due to its effect on OCTN2 in renal tubular cells. As shown in Table 1, steroids such as aldosterone and corticosterone strongly inhibited carnitine transport. Steroids are strong inhibitors of the organic cation transporter family (Koepsell, 1998; Zhang et al, 1998), and because hOCTN2 has a 33.1% similarity with hOCT1 (Zhang et al., 1997) and hOCT2 (Gorboulev et al., 1997), a significant inhibitory effect of steroids on hOCTN2-mediated carnitine transport is not unexpected. Although probenecid and valproate were moderate inhibitors, most of the anionic compounds examined were not inhibitory, therefore hOCTN2 seems to have a preference for cationic rather than anionic moieties. Carnitine deficiency is known to be caused by long-term treatment with valproate (Gerber et al., 1979; Ohtani et al., 1982), pivampicillin (Holme et al., 1989), emetine (Kuntzer et al., 1990), and zidovudine (Dalakas et al., 1994). Although several mechanisms have been postulated to explain the toxicity, the actual mechanism has not yet been clarified at the molecular level. Our present results imply that the competitive inhibition of hOCTN2-mediated carnitine transport by valproate and emetine may be one of the mechanisms of drug-induced carnitine deficiency. Furthermore, other cationic and zwitterionic drugs and some anionic drugs found here to inhbit hOCTN2-mediated carnitine transport may cause drug-induced carnitine deficiency during long-term treatment, especially in tissues such as kidney, heart, and skeletal muscle, where hOCTN2 is highly expressed.

hOCTN1 transports the organic cation TEA in a pH-dependent manner (Tamai et al., 1998). Wu et al. (1998) reported that hOCTN2-mediated TEA transport also shows pH dependence. Furthermore, hOCTN2 has a high similarity (75.8%) with hOCTN1. Accordingly, because it is probable that OCTN2 transports cationic compounds as well as carnitine, we examined whether or not the xenobiotics that inhibit hOCTN2-mediated carnitine transport are substrates of hOCTN2. As expected, uptakes of TEA, pyrilamine, quinidine, verapamil and valproate, all of which showed a significant inhibitory effect on hOCTN2-mediated carnitine transport, were significantly increased in hOCTN2-transfected HEK293 cells (Table 2). However, although MPP, cimetidine, and actinomycin D inhibited hOCTN2-mediated carnitine transport with rather high affinity, they were hardly transported by hOCTN2. This result suggests that good inhibitors of hOCTN2-mediated carnitine transport are not necessarily transported by hOCTN2. Interestingly, the transport of xenobiotics, such as pyrilamine and verapamil, did not show Na+ dependence (Fig. 6). Furthermore, our preliminary experiment showed that a long-chain acylcarnitine, palmitoyl-l-carnitine, was apparently transported in a Na+-independent manner. Although the precise role of Na+ is not clear at present, the transport of organic cations other than carnitine and acetyl-l-carnitine via hOCTN2 seems to occur without any interaction with Na+. Furthermore, it is interesting to compare the substrates between OCTN2 and P-glycoprotein, because some compounds such as quinidine and verapamil are common substrates for these two transporters, and it may be a physiological role for their transporters to facilitate elimination of xenobiotics from the body.

In conclusion, hOCTN2 is physiologically important and multispecific carnitine transporter. Its roles in the transport of acetyl-l-carnitine and other cationic compounds, in combination with the facts that it is inhibited by a wide variety of xenobiotics and shows a wide tissue distribution, imply that hOCTN2 is also of considerable pharmacological and toxicological importance.

Footnotes

  • Send reprint requests to: Akira Tsuji, Ph.D., Department of Pharmacobio-Dynamics, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan. E-mail: tsuji{at}kenroku.kanazawa-u.ac.jp

  • ↵1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan, and by a grant from the Japan Health Sciences Foundation Drug Innovation Project.

  • Received for publication November 6, 1998.

  • Abbreviations:
    jvs
    juvenile visceral steatosis
    TEA
    tetraethylammonium
    MPP
    1-methyl-4-phenylpyridinium
    NMN
    N1-methylnicotinamide
    AZT
    zidovudine
    • Accepted June 3, 1999.

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Na+-Dependent Carnitine Transport by Organic Cation Transporter (OCTN2): Its Pharmacological and Toxicological Relevance

Rikiya Ohashi, Ikumi Tamai, Hikaru Yabuuchi, Jun-Ichi Nezu, Asuka Oku, Yoshimichi Sai, Miyuki Shimane and Akira Tsuji
Journal of Pharmacology and Experimental Therapeutics November 1, 1999, 291 (2) 778-784;
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