Transport of Monoamine Transmitters by the Organic Cation Transporter Type 2, OCT2*

The recently cloned apical renal transport system for organic cations (OCT2) exists in dopamine-rich tissues such as kidney and some brain areas (Gründemann, D., Babin-Ebell, J., Martel, F., Örding, N., Schmidt, A., and Schömig, E. (1997)J. Biol. Chem. 272, 10408–10413). The study at hand was performed to answer the question of whether OCT2 accepts dopamine and other monoamine transmitters as substrate. 293 cells were stably transfected with the OCT2r cDNA resulting in the 293OCT2r cell line. Expression of OCT2r in 293 cells induces specific transport of tritiated dopamine, noradrenaline, adrenaline, and 5-hydroxytryptamine (5-HT). Initial rates of specific3H-dopamine, 3H-noradrenaline,3H-adrenaline, and 3H-5-HT transport were saturable, the K m values being 2.1, 4.4, 1.9, and 3.6 mmol/liter. The corresponding V max values were 3.9, 1.0, 0.59, and 2.5 nmol min−1·mg of protein−1, respectively. 1,1′-diisopropyl-2,4′-cyanine (disprocynium24), a known inhibitor of OCT2 with a potent eukaliuric diuretic activity, inhibited 3H-dopamine uptake into 293OCT2r cells with an K i of 5.1 (2.6, 9.9) nmol/liter. In situ hybridization reveals that, within the kidney, the OCT2r mRNA is restricted to the outer medulla and deep portions of the medullary rays indicating selective expression in the S3 segment of the proximal tubule. These findings open the possibility that OCT2r plays a role in renal dopamine handling.

The inactivation of released hormones and neurotransmitters is essential for chemical signal transduction. The inactivation of monoamine transmitters such as dopamine, noradrenaline, adrenaline, and 5-hydroxytryptamine is brought about by neuronal and non-neuronal (extraneuronal) membrane-bound transport systems. These transport mechanisms actively remove the monoamine transmitters from the extracellular space and thus terminate their biological actions. The primary structures of a variety of sodium-dependent neuronal transport systems for biogenic amines have recently been elucidated (1,2). On the other hand, the molecular correlates of the transporter or transporters responsible for extraneuronal monoamine uptake are still unknown.
The use of a novel class of potent inhibitors of extraneuronal monoamine transport revealed that extraneuronal mechanisms contribute markedly to the inactivation of circulating catecholamines (3). The pharmacological characterization of extraneuronal monoamine transport in tissue culture suggests an unexpected but nevertheless close pharmacological relationship between extraneuronal monoamine transport and the secretion of organic cations through the apical plasma membrane of renal proximal tubule cells (4). Recently, the apical renal transport system for organic cations (OCT2) 1 has been cloned from LLC-PK 1 porcine kidney cells, rat kidney, and human kidney (5)(6)(7). Based on the tissue distribution of the OCT2 mRNA, the hypothesis has been raised that OCT2 is involved in dopamine handling. The OCT2 mRNA was found to exist not only in the kidney but also in dopamine-rich brain areas of the rat (5). The present study was designed to answer the question whether OCT2 accepts monoamine transmitters as substrates.

Construction of a Cell Line That Stably Expresses the Organic Cation
Transporter OCT2r-If not stated otherwise, standard molecular biology techniques were employed (8). For the construction of pcDNA3OCT2r, the cDNA of OCT2r has been released from pBluescript II OCT2r by restriction with HindIII and XhoI and inserted into the corresponding polylinker site of pcDNA3 (Invitrogen, San Diego, CA). pcDNA3 is a eukaryotic expression vector that carries an aminoglycoside resistance cassette. Human embryonic kidney (HEK) 293 cells (ATCC CRL-1573) (9) were transfected by a cationic liposome technique with the Tfx-50 reagent (Promega, Mannheim, Germany) according to the recommendations of the manufacturer. Selection was carried out with the aminoglycoside Geneticin (Life Technologies, Inc.). The 293 OCT2r cells were derived from an isolated transfected cell obtained by single cell cloning. Success of stable transfection of the 293 OCT2r cells was tested by RT-PCR and functional experiments (see "Results"). For control purposes, 293 pcDNA3 cells were constructed that were stably transfected with the empty expression vector pcDNA3. 293 WT refers to wild-type 293 cells.
Transport Assays-The cell lines were grown in surface culture on standard tissue culture plastic materials. After a preincubation period of 30 min at 37°C with buffer A (125 mmol/liter NaCl, 4.8 mmol/liter KCl, 1.2 mmol/liter CaCl 2 , 1.2 mmol/liter KH 2 PO 4 , 1.2 mmol/liter MgSO 4 , 25 mmol/liter HEPES⅐NaOH, pH 7.4, 5.6 mmol/liter D(ϩ)glucose), the cells were incubated with 100 nmol/liter 3 H-labeled substrates in buffer A. Where appropriate, transport inhibitors were present during both the preincubation and incubation periods. Incubation was stopped by rinsing the cells four times with 3 ml of ice-cold buffer * The study was supported by the Deutsche Forschungsgemeinschaft (Un34/19 -1/B2 and SFB601/A4). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ A. Subsequently, the cells were solubilized with 0.1% v/v Triton X-100 (dissolved in 5 mmol/liter Tris⅐HCl, pH 7.4), and radioactivity was measured by liquid scintillation counting. The specific activity of the used radiosubstrates was adjusted to result in uninhibited uptake values of about 500 cpm per culture dish. L(ϩ)-ascorbic acid (1 mmol/ liter) was present in all experiments. Where appropriate, intracellular metabolism via monoamine oxidase (MAO) and/or catechol-O-methyltransferase (COMT) was blocked by 10 mol/liter pargyline alone (in the case of 5-HT) or by 10 mol/liter pargyline and 10 mol/liter U-0521 (in the case of dopamine, noradrenaline, or adrenaline).
In Situ Hybridization-The kidneys were removed from adult Sprague-Dawley rats perfused with 4% paraformaldehyde in phosphatebuffered saline (PBS), pH 7.4, and 18% sucrose solution in PBS. 14-m cryostat sections were mounted on poly-L-lysine-coated slides and postfixed for 20 min in 4% paraformaldehyde. The slides were washed in DEPC-treated water and subsequently in 0.1 M HCl. Next, the sections were acetylated for 20 min in 0.1 M triethanolamine (pH 8.0) containing 0.25% acetic anhydride and, after rinsing in PBS, dehydrated in ethanol (70 and 96%) and air-dried. Digoxigenin-labeling and nonradioactive in situ hybridization was carried out essentially as described elsewhere (11). Digoxigenin-11-UTP labeled sense and antisense RNA probes were prepared from a 1.63-kilobase fragment of the rat OCT2 cDNA and shortened by alkaline hydrolysis to an average length of 250 bases. The sections were hybridized at 42°C for 16 h and washed at 49°C in 1ϫ and 0.5ϫ SSC containing 50% formamide and finally in 0.5ϫ and 0.2ϫ SSC at 20°C. Incubation with alkaline-phosphatase-coupled antidigoxigenin antibodies and the subsequent color reaction were done according to the protocol of the manufacturer (Boehringer Mannheim, Germany).
Protein Determination-Protein was determined by the method of Lowry (12).

Test for Stable Transfection of 293 OCT2r
Cells-Success of stable transfection was tested by RT-PCR. 8 weeks after transfection with pcDNA3OCT2r, total RNA was prepared from the Geneticin-resistant clonal 293 OCT2r cells and, for control purposes, from wild-type 293 cells. With RNA from 293 OCT2r , RT-PCR with primers for OCT2r resulted in the amplification of the expected 795-base pair long PCR product. With RNA from 293 WT cells, there was no amplification product.
Stable transfection was also confirmed by functional testing. Upon incubation with 100 nmol/liter, initial rates of uptake of the prototypical organic cation 3 H-MPP ϩ in 293 OCT2r cells and 293 pcDNA3 control cells amounted to 1.9 and 0.09 pmol min Ϫ1 ⅐mg of protein Ϫ1 , respectively (n ϭ 2).
Uptake of Monoamine Transmitters in 293 OCT2r Cells-Specific uptake of tritiated adrenaline, 5-hydroxytryptamine (5-HT), noradrenaline, and dopamine were analyzed in 293 OCT2r cells and, for control purposes, in 293 pcDNA3 cells. After preincubation of 30 min in buffer A, the cells were incubated for 6 min at 37°C in the presence of one of the labeled amines. Intracellular metabolism of the respective amine was blocked (for details, see "Experimental Procedures"). Specific uptake was defined as that fraction of total uptake which was sensitive to 1 mol/liter decynium22. Decynium22 is a potent inhibitor of OCT2, the K i being 5.6 nmol/liter (4). The expressed transport activity for a given substrate reflects the transport activity induced by the expression of OCT2r, i.e. transport activity in 293 OCT2r cells minus uptake into 293 pcDNA3 control cells. The expression of OCT2r induced specific uptake of all tested monoamine transmitters (Fig. 1). The transport efficiencies ranged from 3.4 Ϯ 0.3 l/(mg of protein⅐min) for adrenaline to 1.0 Ϯ 0.2 l/(mg of protein⅐min) for dopamine.
Transport of dopamine by 293 OCT2r cells was analyzed in more detail. For this purpose, 293 OCT2r cells and 293 pcDNA3 cells were incubated with 100 nmol/liter 3 H-dopamine either in the absence or in the presence of 1 mol/liter decynium22. 3 H-dopamine accumulation increased almost linearly for about 10 min (Fig. 2). The rate constants for total inwardly and outwardly directed 3  librium, the intracellular accumulation of 3 H-dopamine amounted to 8.9 Ϯ 1.3 pmol⅐mg of protein Ϫ1 (n ϭ 15) in 293 OCT2r cells. Based on the known water space of 293 cells (18), this accumulation translates into a factor of intracellular accumulation at equilibrium of 13.
Initial rates of specific 3 H-dopamine transport in 293 OCT2r cells were saturable. The half-saturating concentration was 2.1 (0.7, 5.9) mmol/liter, and the maximal initial rate of 3 H-dopamine uptake amounted to 3.9 Ϯ 0.8 nmol⅐min Ϫ1 ⅐mg of protein Ϫ1 (n ϭ 15). The corresponding Scatchard plot is compatible with simple Michaelis-Menten kinetics ( Fig. 3 and Table I). For comparison, the corresponding kinetic constants for specific 3 H-noradrenaline, 3 H-adrenaline, and 3 H-5-hydroxytryptamine uptake in 293 OCT2r cells were determined (Table I).
The effects of known inhibitors of neuronal, vesicular, and extraneuronal monoamine transport systems were determined to distinguish dopamine transport via OCT2r from known monoamine transport systems. After preincubation of 20 min, 293 OCT2r cells and 293 pcDNA3 cells were incubated for 6 min at 37°C with 100 nmol/liter 3 H-dopamine in the absence and presence of cocaine (1 mol/liter), reserpine (30 nmol/liter), and O-methylisoprenaline (30 mol/liter). The inhibitors were present during both the preincubation and incubation periods but failed to interact significantly with OCT2r (Fig. 4).
The inhibitory potencies of two known inhibitors of OCT2, disprocynium24 and corticosterone, were measured (Table I and Fig. 5). Because nonsaturating concentrations of the labeled substrates (0.1 mol/liter) were used, the IC 50 values are identical with the corresponding K i values. Disprocynium24 and corticosterone inhibited monoamine transport by OCT2r with K i values of about 10 and 500 nmol/liter, respectively.
In Situ Hybridization-Cryostat sections of adult rat kidney were hybridized with digoxigenin-labeled antisense RNA probes transcribed from a 1.63-base pair fragment of OCT2r. OCT2r transcripts were detected in tubule cells of the outer stripe of the medulla as well as in deep portions of the medullary rays (Fig. 6). This pattern indicates exclusive OCT2 mRNA expression in proximal tubule cells of the S3 segment. Proximal tubules of the S1 and S2 segment as well as other structures in renal cortex and medulla such as glomeruli, distal tubules, collecting ducts, and blood vessels were unreactive for the hybridization probe (Fig. 6). On alternate sections, the corresponding sense probe did not produce any detectable signal (not shown).   (n ϭ 12).

DISCUSSION
The primary structure of a new renal transport system for organic cations (OCT2p) has recently been elucidated by molecular cloning from LLC-PK 1 porcine kidney cells (5). OCT2p and OCT2r, the orthologue from rat kidney which has first been reported by Okuda et al. (6), upon heterologous expression in 293 cells, induce saturable transport of prototypical organic cations such as tetraethylammonium and 1-methyl-4-phenylpyridinium (MPP ϩ ). The pharmacological properties clearly demonstrate that OCT2p is identical with the transport system for organic cations that is expressed in the apical but not the basolateral plasma membrane of LLC-PK 1 cells (4, 5). LLC-PK 1 FIG. 6. Localization by in situ hybridization of the OCT2r mRNA in the adult rat kidney. A, intense labeling occurs in proximal tubular profiles located in the outer stripe of the outer medulla and in deeper portions of the medullary rays. The distribution is compatible with the topographical arrangement of the S3 segment of the proximal tubule. B, OCT2r mRNA is expressed in proximal tubular cells of the S3 segment of the deeper part of a medullary ray but not in proximal tubule cells of the S1 and S2 segments located on the right side. cells, a widely used established cell line from porcine kidney (19), display many properties characteristic of proximal tubular epithelial cells (20).
Two recently reported findings open the possibility that OCT2 might be involved in transmembrane transport of monoamines, especially dopamine. Although the pharmacological properties of OCT2 are characteristic of apical renal transport of organic cations, the tissue distribution indicates that the role of OCT2 might go beyond renal secretion of organic cations. RT-PCR reveals that the OCT2r mRNA exists not only in the kidney but is also transcribed in specific brain regions that are especially rich in the monoamine transmitter dopamine such as the nucleus accumbens, striatum, and substantia nigra (5). Moreover, OCT2 is sensitive to corticosterone as well as to the iso-and pseudoisocyanines which represent a novel group of potent inhibitors of extraneuronal monoamine transport (5). It should, however, be noted that, despite the evidence of a close relationship, OCT2 and the extraneuronal monoamine transporter are not identical. It is possible to distinguish these transport systems pharmacologically with O-methylisoprenaline. O-methylisoprenaline inhibits OCT2 about 500 times less potently than the extraneuronal monoamine transporter (5,21).
The present study was designed to clarify whether OCT2 transports dopamine and other monoamine transmitters. To exclude transport-kinetic artifacts known to occur with isolated organs and to minimize the influence of other transport mechanisms that might contribute to monoamine transport, a cell line was created on the basis of human embryonic kidney cells (293 cells) that stably expresses OCT2r (293 OCT2r cells). The success of transfection was documented by RT-PCR and functional testing. For control purposes, 293 cells were also transfected with the empty eukaryotic expression vector pcDNA3 (293 pcDNA3 cells).
Stable transfection of 293 cells with OCT2r induced marked transport of dopamine and other biogenic amines such as 5-HT, noradrenaline, and adrenaline. Initial rates of dopamine transport, mediated by OCT2r, were saturable and sensitive to known inhibitors of the apical renal transport mechanism for organic cations such as decynium22, disprocynium24, and corticosterone (4,5). Cocaine (1 mol/liter), O-methylisoprenaline (30 mol/liter), and reserpine (30 nmol/liter), on the other hand, failed to affect dopamine transport by OCT2r significantly. Cocaine is a known inhibitor of the neuronal dopamine transporter (DAT), the K i being about 0.1 mol/liter (22). O-Methylisoprenaline inhibits the extraneuronal monoamine transporter with a K i of 1.5 mol/liter (21) and reserpine the vesicular monoamine transporters with a K i of 1 nmol/liter (23).
These findings demonstrate that OCT2 has the capability of transporting dopamine and other monoamine transmitters and, hence, open the possibility of OCT2 being involved in dopamine handling in both the kidney and the central nervous system.
Recent evidence from in vivo experiments supports the hypothesis that OCT2 plays a role in renal dopamine handling. In the anesthetized rabbit, intravenous treatment with disprocy-nium24 grossly reduces tubular secretion of dopamine (24). Renal proximal and distal tubule cells express the dopaminesynthesizing enzyme L-aromatic amino acid decarboxylase (25), and proximal tubule cells are known to form and secrete substantial amounts of dopamine (26). Several lines of evidence support the concept that dopamine acts as an intrarenal natriuretic hormone, possibly through an indirect modulation of the sodium/potassium-ATPase activity in tubular cells (27).
Disprocynium24 belongs to the new group of potent inhibi-tors of OCT2 and the related extraneuronal monoamine transporter (4,17). It is, in fact, the most potent inhibitor of OCT2 reported so far. The K i for the inhibition of OCT2r has been shown to be as low as 2.4 nmol/liter (5). This figure fits in with the present results that reveal a K i of 5.1 nmol/liter of dispro-cynium24 for the inhibition of dopamine transport by OCT2. Interestingly enough, disprocynium24 does not only block tubular secretion of dopamine but induces marked spill-over of renal dopamine into the systemic circulation (24). This finding is compatible with the concept that dopamine from proximal tubule cells is normally secreted by OCT2 through the apical plasma membrane into the lumen of the tubule. When OCT2 is blocked, intracellular dopamine rises and significant spill-over into the peritubular fluid and, subsequently, systemic circulation occurs. The localization of the OCT2r mRNA in the kidney fits in with this concept. Our results indicate that high levels of the OCT2r mRNA exists in proximal tubule cells of the S3 segment. Expression of OCT2h, a human homologue of OCT2r, has been reported in the apical plasma membrane of distal tubule cells. But there is also evidence for OCT2h to exist in proximal tubule cells (7). There is ample evidence that renal dopamine is involved in the regulation of sodium excretion (27) and, surprisingly enough, in the anaesthetized rat, disprocynium24 does not only block dopamine excretion but acts as a high ceiling eukaliuric diuretic and natriuretic (28). The exact mechanism of this new pharmacological effect of disprocynium24 has not yet been clarified. An indirect activation of dopamine D1 and D2 receptors, however, does not seem to be involved since pretreatment with the dopamine receptor blockers SCH23390 and S(Ϫ)-sulpiride fails to affect the diuretic action of disprocynium24 (28).
The recently cloned apical renal transport systems for organic cations OCT2 exists in proximal tubule cells and in dopamine-rich brain areas. For the first time, a cell line that stably expresses OCT2 has been created (293 OCT2r cells) and used to analyze the substrate specificity with special emphasis on monoamine transmitters. The finding that OCT2 accepts dopamine as a substrate opens the possibility that this transporter is involved in dopaminergic signal transduction. Together with recently published in vivo data, the findings at hand suggest that OCT2 plays an important role in renal dopamine handling and might be involved in the potent eukaliuric diuretic action of disprocynium24.