Experimental Procedures

Materials.

[1-¹⁴C]TEA bromide (2.4 mCi/mmol),p-[glycyl-1-¹⁴C]aminohippuric acid (53.1 mCi/mmol), and [N-methyl-³H]verapamil hydrochloride (78.6 Ci/mmol) were purchased from New England Nuclear (Boston, MA).l-[Methyl-³H]carnitine hydrochloride and [pyridinyl-5-³H]pyrilamine (28 Ci/mmol) were purchased from Amersham Pharmacia Biotech UK, Ltd. (Buckinghamshire, England). [9-³H]Quinidine (15 Ci/mmol) was from ARC Inc. (St. Louis, MO). All other chemicals were purchased from Sigma (St. Louis, MO) and Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Transport Experiments in Xenopus Oocytes.

Oocytes from Xenopus laevis were obtained by manual dissection in medium A (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 5 mM HEPES adjusted to pH 7.6 with NaOH) and defolliculated in modified Barth’s solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.82 mM MgSO₄, 0.33 mM Ca(NO₃)₂, 0.41 mM CaCl₂, 100 U/ml penicillin G, 100 mg/ml streptomycin, and 10 mM HEPES adjusted to pH 7.5 with NaOH).

Human OCTN1 cDNA (Tamai et al., 1997) was subcloned into theHindIII/EcoRI sites of the expression vector pcDNA3 (Invitrogen, San Diego, CA). The construct pcDNA3/OCTN1 was then linearized with EcoRI and used for in vitro transcription after capping. cRNA was dissolved in water at a concentration of 0.2 μg/μl and injected (50 nl) into oocytes, which were used for the assay of transport activity after cultivation for 3 or 4 days. To measure uptake of organic cations, oocytes (5–10/individual time point or condition) were incubated in 250 μl of transport medium (100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES/NaOH, pH 7.5) containing a radiolabeled compound at 25°C. The uptake was terminated by washing the oocytes three times with 15 ml of ice-cold transport buffer. Washed oocytes were transferred to vials containing 100 μl of 5% SDS to be solubilized and the associated radioactivity was measured with a liquid scintillation counter. When transport was measured at an acidic or alkaline pH, 2-(N-morpholino) ethanesulfonic acid, or Tris was used instead of HEPES to maintain the desired pH, respectively.

Uptake and Efflux Experiments by Transient Expression in HEK293 Cells.

The construct pcDNA3/OCTN1 was used to transfect HEK293 cells (Japanese Cancer Research Resources Bank, Tokyo, Japan) according to the calcium phosphate precipitation method. HEK293 cells were routinely grown in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (Gibco, Grand Island, NY), penicillin, and streptomycin in a humidified incubator at 37°C under 5% CO₂. After 24 h cultivation of HEK293 cells in 10-cm dishes, pcDNA3/OCTN1 or pcDNA3 vector alone was transfected by adding 10 μg of the plasmid DNA per dish. At 48 h after transfection, the cells were harvested and suspended in transport medium containing 125 mM NaCl, 4.8 mM KCl, 5.6 mMd-glucose, 1.2 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, and 25 mM HEPES (pH 7.4).

In uptake experiments, cell suspension and a solution of a radiolabeled test compound in the transport medium were each incubated at 37°C for 20 min, then transport was initiated by mixing them. At appropriate times, 200-μl aliquots of the mixture were withdrawn and the cells were separated from the transport medium by centrifugal filtration through a layer of mixture of silicon oil and liquid paraffin with a density of 1.03. Each cell pellet was solubilized in 3 N KOH, neutralized with HCl, and the associated radioactivity was measured by means of a liquid scintillation counter. In the [¹⁴C]TEA efflux assay, HEK293 cells were preloaded with 0.5 μCi of [¹⁴C]TEA at 37°C for 10 min, then pelleted by centrifugation at 10,000 rpm for 10 s, and efflux was initiated by resuspending the cells in transport medium. At the indicated time, efflux was terminated by centrifugal filtration as described above. Cellular protein content was measured according to the method of Bradford (1976) using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) and BSA as the standard.

Data Analysis.

All data were expressed as mean ± S.E.M. and statistical analysis was performed by the use of Student’st test. The criterion of significance was taken to beP < .05.

Results

Expression of [¹⁴C]TEA Uptake inXenopus Oocytes Injected with Human OCTN1 cRNA.

To confirm the expression of functional organic cation transport activity of human OCTN1, we investigated the OCTN1-mediated [¹⁴C]TEA transport in a Xenopusoocyte heterologous expression system. The uptake of [¹⁴C]TEA in OCTN1 cRNA-injected oocytes was significantly higher than that in water-injected oocytes on day 2 and was further increased on days 3 and 4 after injection of the cRNA (Fig.1). Subsequent uptake studies were carried out more than 3 days after cRNA injection. Although the data are not shown, uptake of [¹⁴C]TEA] byXenopus oocytes was linearly increased till 90 min and saturable with apparent K _m andV _max values of 0.195 ± 0.033 mM and 18.5 ± 1.45 pmol/60 min/oocyte, respectively.

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Figure 1

Effect of cultivation time of oocytes after cRNA injection on uptake of [¹⁴C]TEA for 60 min. Uptake of [¹⁴C]TEA (60 μM) in OCTN1 cRNA (10 ng)-injected (●) and water (50 nl)-injected (○) oocytes was measured at 25°C in transport medium (pH 7.4). Results are shown as means ± S.E. of 5 to 10 oocytes. When S.E. was smaller than symbols, it was not shown.

Effect of Membrane Potential on [¹⁴C]TEA Uptake inXenopus Oocytes.

We examined pH dependence of TEA uptake by OCTN1 expressed in Xenopus oocytes. When the pH in the transport medium was acidic (pH 6.0), OCTN1-mediated uptake of [¹⁴C]TEA was decreased to about 40% of those at neutral and alkaline pH, whereas no significant change in the uptake of [¹⁴C]TEA was observed in water-injected oocytes. Furthermore, comparable uptakes were observed at neutral and alkaline pH (data not shown). The observation was comparable with that observed in TEA transport by OCTN1 expressed in HEK293 cells (Tamai et al., 1997).

Figure 2 shows the effect of membrane potential on [¹⁴C]TEA uptake by OCTN1 examined by cRNA-injected Xenopus oocytes. The uptake of [¹⁴C]TEA was not significantly altered upon replacement of Na⁺ with K⁺. Accordingly, a lack of membrane potential dependent transport of TEA by OCTN1 was suggested.

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Figure 2

Effect of membrane potential on [¹⁴C]TEA uptake in OCTN1 cRNA-injected oocytes. Uptakes of [¹⁴C]TEA (60 μM) in OCTN1 cRNA (10 ng)-injected (filled columns) and water (50 nl)-injected (open columns) oocytes were measured for 60 min at 25°C in the presence or absence of Na⁺ in transport medium (pH 7.4). Results are shown as means ± S.E. of 5 to 10 oocytes. When S.E. was smaller than symbols, it was not shown.

Effect of Extracellular pH on Efflux of [¹⁴C]TEA in HEK293 cells.

Because it is important to know the driving force for TEA transport by OCTN1 to differentiate the physiological role on this transporter from those of the previously known OCT1 and OCT2, we further examined the effect of extracellular pH on TEA efflux from OCTN1-transfected HEK293 cells (Table 1). Efflux of [¹⁴C]TEA was initiated by incubating the cells preloaded with 150 μM [¹⁴C]TEA in the fresh medium at various pHs, and the efflux was evaluated from the remaining amount of [¹⁴C]TEA in the cells. Table 1 shows the time course of the amount of remaining [¹⁴C]TEA in the cells as the percentage of initial. The remaining amount of [¹⁴C]TEA at acidic pH 6.0 was minimum and increased with the increase of pH up to pH 8.0 over 5 min, demonstrating an enhanced efflux at acidic pH.

Inhibition of [¹⁴C]TEA Uptake by Various Cationic and Anionic Compounds in HEK293 Cells.

The effect of various endogenous compounds and xenobiotics on the OCTN1-mediated [¹⁴C]TEA uptake in HEK293 cells is shown in Table 2. Various cationic compounds, such as choline, clonidine, cimetidine, nicotine, procainamide, pyrilamine, quinine, quinidine, and verapamil, had significant inhibitory effects (p < .05). Furthermore, zwitterionic compounds such as l- and d-carnitine and aminocephalosporin, cephaloridine and quinolone antibacterial agents, levofloxacin, and ofloxacin, were also significantly inhibitory, whereas other organic cations, such as NMN, guanidine, putrescine, spermine, and spermidine were not inhibitory. Cationic amino acids (l-arginine and l-lysine) and anionic compounds (p-aminohippurate and benzylpenicillin) were not inhibitory either.

Transport of Various Organic Cationic Compounds.

The substrate selectivity of OCTN1 was assessed by measuring the uptake of radiolabeled compounds by Xenopus oocytes expressing OCTN1, because HEK293 cells exhibited significantly high background level of uptake. As shown in Fig. 3, although background uptakes were still rather high because of the binding, nonmediated diffusion and/or native transport activity ofXenopus oocytes, uptakes of [³H]quinidine, [³H]pyrilamine, [³H]verapamil, andl-[³H]carnitine, as well as [¹⁴C]TEA, by OCTN1 cRNA-injected oocytes were apparently increased in comparison with those by water-injected oocytes (p < .05). In contrast, the uptake of [¹⁴C]benzylpenicillin by OCTN1 cRNA-injected oocytes was not increased.

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Figure 3

Transport of various organic cations by OCTN1. Uptakes of [¹⁴C]TEA (60 μM),l-[³H]carnitine (10 nM), [³H]quinidine (65 nM), [³H]pyrilamine (35 nM), [³H]verapamil (10 nM), and [¹⁴C]benzylpenicillin (20 μM) in water-injected (open columns) or OCTN1 cRNA-injected (closed columns) oocytes were measured for 60 min at 25°C in transport buffer (pH 7.5). Each value represents means ± S.E. of 7 oocytes. When S.E. was smaller than symbols, it was not shown. *Significantly different from the uptake by water-injected oocytes by Student’s t test (p < .05).

Effect of Extracellular TEA and Quinidine on Efflux of [¹⁴C]TEA in HEK293 Cells.

To examine the specificity and direction of TEA transport, the effect of substrate/inhibitor of OCTN1 on the efflux of [¹⁴C]TEA from HEK293 cells was examined. When unlabeled TEA was included in the external medium, the intracellular [¹⁴C]TEA decreased faster than control, whereas in the presence of extracellular quinidine the remaining amount of [¹⁴C]TEA was kept high (Table 1). Effects of TEA and quinidine are explained by the enhancement of [¹⁴C]TEA efflux by an exchange transport with unlabeled TEA and inhibition of [¹⁴C]TEA efflux by quinidine, which is taken up by the HEK293 cells during efflux study rather than the enhancement by exchange transport, respectively. These observations suggest that efflux of [¹⁴C]TEA from the HEK293 cells is mediated by OCTN1 in a cation exchange mechanism.

Discussion

Recently, we cloned a novel organic cation transporter, human OCTN1, which is strongly expressed in kidney and resulted in pH-dependent TEA uptake when expressed in HEK293 cells (Tamai et al., 1997). In the present study, we further investigated the precise functional properties of organic cation transport by OCTN1 using two gene expression systems, Xenopus oocytes and HEK293 cells.

OCTN1 cRNA-injected oocytes showed significantly enhanced transport of [¹⁴C]TEA as compared with water-injected oocytes. The K _m for OCTN1-mediated TEA uptake obtained in Xenopus oocytes (0.195 mM) was close to those of the previously characterized OCTN1-mediated TEA uptake in HEK293 cells (0.436 mM; Tamai et al., 1997) and in isolated brush-border membrane vesicles from rat and rabbit (0.15 ∼ 0.8 mM; Takano et al., 1984; Wright and Wunz, 1987; Maeda et al., 1993). In addition, similar pH dependence in [¹⁴C]TEA uptake by OCTN1 cRNA-injected oocytes to that by OCTN1-transfected HEK293 cells was observed (Tamai et al., 1997). However, the uptake of [¹⁴C]TEA in OCTN1 cRNA-injected oocytes was not significantly affected upon replacement of Na⁺with K⁺ (Fig. 2). The absence of potassium replacement effect is quite distinct from the apparent pH- and membrane potential-dependent transport as observed in OCT3 (Kekuda et al., 1998) and, therefore, suggests that the apparent pH-dependent transport of TEA by OCTN1 is not accounted for by the change of membrane potential. Moreover, [¹⁴C]TEA efflux from OCTN1-transfected HEK293 cells was stimulated by an increase in the proton concentration of the external medium (Table 1). Accordingly, the transport of TEA by OCTN1 is presumably driven directly with a movement of proton but not with an inside-negative membrane potential difference as discussed in our previous observations obtained in a mammalian gene expression system (Tamai et al., 1997). The absence of further increase of TEA uptake at an alkaline pH compared with neutral pH is apparently comparable with the observation in TEA uptake by renal brush-border membrane vesicles (Maegawa et al., 1988). pH dependence observed in OCTN1-mediated TEA uptake might be ascribed to at least two underlying mechanisms, including activation by outwardly proton gradient and by the presence of functionally optimal protonated form of the transporter protein.

To determine the specificity of OCTN1, we examined the effect of various compounds on the initial uptake of [¹⁴C]TEA and the uptake of several organic cations that exhibited inhibitory effects on TEA uptake (Table 2 and Fig. 3). The initial uptake of [¹⁴C]TEA was significantly inhibited by a number of classical inhibitors on organic cation transport (cimetidine, procainamide, quinine, quinidine, and choline), other organic cationic compounds (clonidine, nicotine, pyrilamine, and verapamil) and zwitterionic compounds such asl-carnitine, aminocephalosporin (cephaloridine), and quinolone antibacterial agents (levofloxacin and ofloxacin), whereas p-aminohippuric acid and benzylpenicillin (typical substrates of organic anion transporter), l-arginine,l-lysine, and the endogenous polyamines spermine, spermidine, and putrescine had no inhibitory effect (Table 2). Moreover, we observed the increased uptake of several radiolabeled cationic compounds by expression of OCTN1 in Xenopusoocytes, suggesting that these cationic compounds as well as TEA are transported by OCTN1 and that the transporter may be multispecific. These data are consistent with previous observations on proton/organic cation transport in renal brush-border membrane vesicles in the human and other species. The absence of inhibition by guanidine suggested that OCTN1 is a pH-dependent transporter that has specificity for TEA and is different from the guanidine transporter in the renal proximal tubule.

TEA transport via OCTN1 was not inhibited by 5 mM NMN, suggesting a low affinity of NMN for OCTN1, although many previous studies have demonstrated that TEA and NMN appear to share the same proton/organic cation transport system (Zhang et al., 1998). However, Koepsell (1998)suggested that the affinity of the proton/TEA antiporter for NMN is low in renal proximal tubules, where a K _ivalue of 8.3 mM for NMN was obtained (David et al., 1995). The difference among these results may be ascribed to experimental artifact, species differences, or the existence of multiple cation antiporters. Because of its broad substrate specificity, OCTN1 is likely to play a significant role as a xenobiotic transporter and may serve as a pharmacologically important multispecific cationic drug transporter.

Efflux of [¹⁴C]TEA was accelerated in the presence of the unlabeled TEA in the external medium, whereas quinidine caused a reduction of the efflux rate (Table 1). As clearly shown in Table 1 and Fig. 3, quinidine as well as TEA is a substrate of OCTN1 and has strong inhibitory potency to [¹⁴C]TEA transport by OCTN1. The uptake efficiency of TEA by OCTN1 expressed inXenopus oocytes is about 32 times less than that of quinidine when evaluated by the cell per medium concentration ratios calculated from the results shown in Fig. 3. Accordingly, it is expected that quinidine added in the external medium accumulates extensively and rapidly into the intracellular space during the efflux of [¹⁴C]TEA and competitively inhibits efflux of [¹⁴C]TEA at the internal side of the cells. On the other hand, TEA accumulation does not occur in the extensive amount during efflux study and it hardly inhibits the efflux of [¹⁴C]TEA from the cells. Accordingly, an enhancement of efflux by the unlabeled TEA may represent countertransport effect, suggesting that OCTN1-mediated TEA transport is symmetrical and is explained by cation/cation exchange mechanism.

Wu et al. (1998) and we (Tamai et al., 1998) have successfully cloned OCTN2, an isoform of OCTN1. Wu et al. (1998) reported a pH-dependent TEA transport via OCTN2, whereas we found that OCTN2 predominantly transports L-[³H]carnitine in a sodium-dependent manner in OCTN2-transfected HEK293 cells, with only a low activity of TEA transport (Tamai et al., 1998). Furthermore, physiological importance of OCTN2 as the carnitine transporter was demonstrated in our recent study on carnitine-deficient patients and mice (Nezu et al., 1999). In the present study, [¹⁴C]TEA transport via OCTN1 was significantly inhibited by l- and d-carnitine andl-carnitine was transported by OCTN1. Accordingly, the common physiological role of OCTN1 and OCTN2 might be transport of carnitine in several tissues, although carnitine transport by OCTN1 will need to be functionally characterized further.

In conclusion, OCTN1 was demonstrated to be a multispecific, bidirectional, and pH-dependent organic cation transporter that is presumably energized by proton antiport mechanism in renal epithelial cells, although its subcellular localization is not clear at present. In addition, the broad substrate specificity of OCTN1 suggests a crucial role of the transporter in facilitating the elimination of xenobiotics. Furthermore, expression of OCTN1 in oocytes and HEK293 cells is expected to be useful for the development of in vitro assay systems for drug elimination and drug/drug interaction studies.