Departments of 1 Biochemistry and Molecular Biology and 2 Medicine and 3 Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912
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ABSTRACT |
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We examined in this study the expression of the potential-sensitive organic cation transporter OCT3 in the kidney. A functionally active OCT3 was cloned from a mouse kidney cDNA library. The cloned transporter was found to be capable of mediating potential-dependent transport of a variety of organic cations including tetraethylammonium. This function was confirmed in two different heterologous expression systems involving mammalian cells and Xenopus laevis oocytes. We have also isolated the mouse OCT3 gene and deduced its structure and organization. The OCT3 gene consists of 11 exons and 10 introns. In situ hybridization studies in the mouse kidney have shown that OCT3 mRNA is expressed primarily in the cortex. The expression is evident in the proximal and distal convoluted tubules. The expression of OCT3 in human kidney was confirmed by RT-PCR. We have also cloned OCT3 from human placenta and human kidney. Human OCT3 exhibits 86% identity with mouse OCT3 in amino acid sequence. Human OCT3 was found to transport tetraethylammonium and a variety of other organic cations. The transport process was electrogenic. We conclude that OCT3 is expressed in mammalian kidney and that it plays an important role in the renal clearance of cationic drugs.
cationic drugs; renal excretion; in situ hybridization; gene structure
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INTRODUCTION |
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MAMMALIAN KIDNEY plays an important role in the elimination of a variety of structurally diverse xenobiotics. Specific transport mechanisms exist in the brush-border membrane and in the basolateral membrane of the kidney tubular cells, which work together in a coordinated manner to mediate the vectorial transport of positively charged xenobiotics from the blood into the tubular lumen (19, 20, 22, 25). Cationic xenobiotics enter the tubular cells from the blood via potential-sensitive organic cation transporters located in the basolateral membrane and are eliminated from the cells by organic cation/H+ antiporters located in the brush-border membrane. In recent years, considerable progress has been made in the elucidation of the molecular nature of some of these transporters (12). To date, two different potential-sensitive organic cation transporters (OCTs) have been cloned and characterized from mammalian kidney. Grundemann et al. (5) cloned the first organic cation transporter, designated OCT1, from rat kidney that transports diverse organic cations using membrane potential as the driving force. In situ hybridization in rat kidney has demonstrated the expression of OCT1-specific mRNA transcripts in proximal tubules but not in distal tubules, glomeruli, and collecting ducts (5). Interestingly, the tissue distribution pattern of OCT1 is dependent on the animal species studied. In rat, OCT1 is expressed most abundantly in kidney, moderately in liver, and at very low levels in intestine (5). In rabbit, OCT1 expression is highest in liver, although mRNA transcripts are detectable at significant levels in kidney and intestine (23). In contrast, the expression of OCT1 is limited primarily to liver in humans (4, 30). Okuda et al. (15) cloned the second organic cation transporter OCT2, also from rat kidney. Subsequent studies with OCT2 cloned from human kidney showed that OCT2 is also a potential-dependent organic cation transporter (4). The expression of OCT2 appears to be primarily restricted to kidney in rat as well as in humans (4, 15). Thus, of the two potential-sensitive organic cation transporters that have been cloned and characterized, only OCT2 is likely to play a role in the renal elimination of cationic xenobiotics in humans. Interestingly, the expression of OCT2 in human kidney is restricted to distal convoluted tubules (4). This observation raises questions as to the functional relevance of OCT2 to renal clearance of cationic xenobiotics, because only the proximal tubule is considered to play the principal role in organic cation transport in kidney (19, 20, 22, 25). The absence of expression of OCT1 and the restriction of OCT2 expression to distal tubules in human kidney strongly suggest that organic cation transporters, hitherto not yet identified, are responsible for renal handling of organic cations in humans.
A third organic cation transporter, called OCT3, was recently cloned and characterized in our laboratory (10, 27). OCT3 was cloned from rat placenta. The expression of OCT3 is most abundant in this tissue. Northern blot analysis has however, indicated that rat kidney expresses moderate levels of mRNA transcripts that hybridize to OCT3 cDNA. This raises the possibility that OCT3 may be expressed in mammalian kidney and play a role in the renal elimination of cationic organic compounds. In this report, we provide unequivocal evidence for the expression of OCT3 in mammalian kidney. This was done by successful cloning of OCT3 from mouse kidney and its functional identification as a potential-sensitive organic cation transporter. Furthermore, we have performed in situ hybridization to determine the expression pattern of OCT3 mRNA in the mouse kidney that shows abundant expression of this organic cation transporter in the proximal as well as distal convoluted tubules. In addition, we have also cloned the human OCT3, established its functional identity as a potential-sensitive polyspecific organic cation transporter, and demonstrated unequivocally its expression in human kidney. These studies strongly suggest that OCT3 participates in the renal handling of cationic xenobiotics in humans and other mammals.
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MATERIALS AND METHODS |
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Materials. [14C]tetraethylammonium (TEA) bromide, [14C]guanidine, and [3H]1-methyl-4-phenylpyridinium (MPP) iodide were purchased from American Radiolabeled Chemicals (St. Louis, MO). The human cell lines HeLa (a cervical carcinoma cell line), JAR (a choriocarcinoma cell line), and Caco-2 and HT-29 (intestinal cell lines) were obtained from the American Type Culture Collection (Rockville, MD). The human choriocarcinoma cell line BeWo, the human breast cancer cell line MCF-7, and the human kidney proximal tubular cell line HKPT were kindly provided by Dr. Aaron J. Moe (Washington University, St. Louis, MO), Dr. Jeffery Moscow (National Cancer Institute, Bethesda, MD), and Dr. Ullrich Hopfer (Case Western Reserve University, Cleveland, OH), respectively. Human retinal pigment epithelial (HRPE) cells were originally provided by Dr. M. A. Del Monte (University of Michigan, Ann Arbor, MI) and have been in use in our laboratory for several years (8). Cell culture media and Lipofectin were from Life Technologies (Gaithersburg, MD). Restriction enzymes were from Promega (Madison, WI). Magna nylon transfer membranes were from Micron Separations (Westboro, MA).
Isolation of the mouse OCT3 cDNA.
A mouse kidney cDNA library (21) was screened under
low-stringency conditions (11) using an Nco
I/Sac II fragment (1.4 kb) of rat OCT3 cDNA
(10) as a probe. The cDNA fragment was labeled with
[-32P]dCTP using the Ready-to-go oligolabeling kit
(Pharmacia). Hybridization was carried out for 20 h at 60°C in a
solution containing 5× SSPE (1× SSPE = 0.15 M NaCl, 10 mM
NaH2PO4, and 1 mM EDTA), 5× Denhardt solution,
0.5% SDS, and 100 µg/ml denatured salmon sperm DNA. Posthybridization washing involved extensive washes with 3× SSPE/0.5% SDS at room temperature. Positive clones were identified, and the
colonies were purified by secondary or tertiary screening. The positive
clones were initially analyzed by sequencing at their 5' ends and by
restriction fragment analysis. OCT3 clones were identified among the
positive clones by comparing the partial amino acid sequence of these
clones deduced from their 5' end sequence with rat OCT3 amino acid
sequence, and the longest of the OCT3 clones was chosen for
further structural and functional analysis.
Analysis of the mouse OCT3 gene. A bacterial artificial chromosome (BAC) library of the mouse (129 SvJ strain) genomic DNA was screened using the full-length mouse OCT3 cDNA as the probe. This screening service was provided by Genome Systems (St. Louis, MO). The positive BAC genomic clone was digested with Hind III, and the fragments were electrophoresed and then subjected to Southern analysis with mouse OCT3 cDNA as the probe. The hybridization-positive fragments were subcloned into pSPORT for sequence determination. The regions between the hybridization-positive fragments were identified by PCR using the Not I-linearized BAC genomic clone as the template and using primers designed on the basis of the nucleotide sequence of the hybridization-positive fragments. The PCR products were subcloned into pGEM-T vector for sequence analysis.
Isolation of the human OCT3 cDNA. This was done by screening a human placental cDNA library (1, 18) using a Nco I/Sac II fragment (1.4 kb) of the rat OCT3 cDNA as the probe. Several positive clones were identified, and sequence analysis indicated that none of them contained the full-length cDNA. The longest clone had a 2.8-kb cDNA that showed significant homology to rat and mouse OCT3 cDNAs. By comparison with the sequences of rat OCT3 and mouse OCT3, it was evident that the partial cDNA lacked ~500 bp at the 5' end. Grundemann et al. (6) have recently cloned a corticosterone-sensitive extraneuronal monoamine transporter (EMT) from Caki-1 cells, a human kidney carcinoma cell line. Amino acid sequence analysis indicated that EMT is the human homolog of rat and mouse OCT3. Nucleotide sequence alignment between EMT and the longest partial clone of human OCT3 showed that they were 100% identical, with the partial clone starting at nucleotide position 566 of the EMT sequence. The missing part of the human OCT3 cDNA was then obtained by RT-PCR using mRNA from the HKPT cell line (9) and spliced into the partial clone to generate the full-length cDNA. The primers were 5'-GGCGGGCGCACCATGCCCTCCTTC-3' (sense primer corresponding to the nucleotide position 16-39 in EMT) and 5'-CACAATCCTCCTTTGTTTCGAACC-3' (antisense primer corresponding to the nucleotide position 730-753 in EMT). RT-PCR was performed using HKPT cell mRNA, and the resultant PCR product (738 bp in size) was subcloned into pGEM-T vector. The identity of the cDNA insert was confirmed by sequencing. The full-length human OCT3 cDNA was then generated from the partial clone and the RT-PCR product using the restriction enzymes EcoR I and BstB I.
DNA sequence determination and analysis. Sequencing of the cDNAs was done by the dideoxy chain termination method with an automated Perkin-Elmer Applied Biosystems 377 Prism DNA sequencer. The sequence was analyzed using the GCG sequence analysis software package GCG version 7.B (Genetics Computer Group, Madison, WI).
Functional expression of mouse and human OCT3 cDNAs in mammalian cells. The cloned mouse and human OCT3 cDNAs were oriented in the pSPORT vector in such a way that their expression was under the control of the T7 promoter. The cDNAs were heterologously expressed in HRPE cells by vaccinia virus expression system as described previously (27). Transport measurements were made at room temperature using radiolabeled substrates. The transport buffer was composed of 25 mM Tris-HEPES (pH 8.5), supplemented with 140 mM N-methyl-D-glucamine chloride, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, and 5 mM glucose. Transport buffers of varying pH were prepared by appropriately mixing 25 mM Tris-HEPES buffer, pH 8.5, and 25 mM MES-Tris buffer, pH 5.5. The buffers were supplemented with N-methyl-D-glucamine chloride, KCl, CaCl2, MgSO4, and glucose as described earlier. These studies were done with HRPE cells cultured in 24-well culture plates. Transport was initiated by adding transport buffer (250 µl) containing radiolabeled substrate to each well. After incubation for a desired time, transport was terminated by aspiration of the buffer followed by two washes with 2 ml of ice-cold transport buffer. The cells were then solubilized with 0.5 ml of 1% SDS in 0.2N NaOH and transferred to vials for quantitation of radioactivity associated with the cells. HRPE cells transfected with vector alone under similar conditions served as control. In experiments dealing with saturation kinetics, data were analyzed by nonlinear regression and confirmed by linear regression.
Functional expression of mouse and human OCT3s in Xenopus laevis oocytes. Mature (stages V-VI), defolliculated oocytes from X. laevis (Nasco, Fort Atkinson, WI) were selected and maintained at 18°C in modified Barth's medium (16) with 10 mg/l gentamycin. Oocytes were injected 1 day after isolation with 50 ng of mouse or human OCT3 cRNA. The transport function of the heterologously expressed OCT3 was studied either by measuring the uptake of radiolabeled organic cations (mouse OCT3) or by monitoring organic cation-induced currents under voltage-clamp conditions (human OCT3). Uptake of radiolabeled substrates into oocytes was measured in a 24-well microtiter plate as described previously (2). Uptake measurements were made at room temperature in oocytes 5-8 days after cRNA injection. The influence of membrane potential on the uptake was studied as described previously (5, 10) by changing the concentration of K+ in the uptake buffer or by adding Ba2+, a K+ channel blocker, to the uptake buffer. Organic cation-induced currents were monitored by using a two-microelectrode voltage-clamp technique as previously described for rat OCT3 (10, 27). Human OCT3-expressing oocytes were perifused with a Na+-free buffer (5 mM HEPES-Tris, 96 mM N-methyl-D-glucamine chloride, 2 mM KCl, 1.8 mM CaCl2, and 1 mM MgCl2, pH 7.5) containing 10 mM TEA or 1-methyl-4-phenylpyridinium (MPP), and the steady-state TEA- and MPP-induced currents were monitored.
The mouse and human OCT3 cRNAs were synthesized using the Not I-linearized pSPORT-cDNA constructs as the template. Transcription was carried out with T7 RNA polymerase in the presence of ribonuclease inhibitor and RNA cap analog. The mMESSAGE mMACHINE kit (Ambion, Austin, TX) was employed for this purpose.Northern blot analysis. The tissue distribution of human OCT3 mRNA was investigated by Northern blot analysis using a commercially available hybridization-ready human multiple tissue Northern blot (1) containing size-fractionated mRNAs from 12 different tissues (Clontech). This blot was hybridized sequentially with 32P-labeled human OCT3 cDNA and 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. The expression of OCT3 mRNA in human cell lines was also investigated by Northern blot analysis using mRNA from HKPT, Caco-2, HT-29, JAR, BeWo, HeLa, and MCF-7 cells. This blot containing size-fractionated mRNAs from different cell lines has been used previously for the analysis of distribution of OCTN2 mRNA (28). The human OCT3 cDNA probe was a ~1.9-kb fragment released from the full-length human OCT3 cDNA by digestion with Bgl II/Kpn I. This fragment comprised of the COOH-terminal one-third of the coding region and most of the 3'-untranslated region of the cDNA. This probe showed no significant homology to human OCT1 or human OCT2 at the level of nucleotide sequence. Hybridization and posthybridization washings were done under high-stringency conditions.
RT-PCR analysis of OCT3 mRNA expression in human kidney and HKPT cell line. The expression of OCT3 mRNA in human kidney and HKPT cell line was examined by RT-PCR and restriction analysis of the RT-PCR products. The human OCT3-specific primers used in this analysis were 5'-TGTAAATGTGGCAGGAATAA-3' (sense) and 5'-GTGAATAAAGGGTGAATGTA-3' (antisense). These primers enclosed the region containing the nucleotide sequence 1648-3097 of the human OCT3 cDNA. mRNA samples from human kidney and HKPT cells were used for RT-PCR. Human placental mRNA was used as a positive control. The identity of the RT-PCR products was established by restriction analysis using three enzymes (Hind III, Nco I, and Pvu II).
In situ hybridization.
Adult mouse kidneys were collected and immediately frozen in liquid
nitrogen. Unfixed 12-µm serial sections were prepared on a cryostat,
mounted on 2% 3-aminopropyltriethoxysilane-coated slides, air-dried
for 10 min, and stored at 70°C until required. Nonradioactive in
situ hybridization using digoxigenin UTP-labeled riboprobes (sense and
antisense) was performed on tissue sections as described previously
(27, 29). Cryostat sections were fixed in 4%
paraformaldehyde in 0.1 M PBS, pH 7.3, at 4°C for 20 min, washed, and
permeabilized with proteinase K (10 µg/ml in PBS containing 0.1%
Tween 20) for 10 min at room temperature. Sections were then refixed in
4% paraformaldehyde in PBS, prehybridized in 5× SSC (1× SSC = 0.15 M NaCl, 15 mM sodium citrate) for 1 h at 65°C in 50%
formamide, and incubated with probes for 16 h at 65°C. Sections were then washed twice in 2× SSC at room temperature, twice in 1× SSC
at 55°C, and twice in 0.1× SSC at 37°C. For immunologic detection
of the probe, sections were incubated for 2 h with
anti-digoxigenin antibody coupled to alkaline phosphatase. Sections
were washed, and the color was developed with the chromogenic substrate
nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate provided in
the kit. Separate, nonhybridized sections were stained with hematoxylin and eosin. For the preparation of the mouse OCT3-specific probes, a
BamH I/EcoR I fragment (0.9 kb) of the mouse OCT3
cDNA was subcloned into pSPORT vector. Antisense and sense riboprobes
were synthesized with SP6 RNA polymerase and T7 RNA polymerase,
respectively, after linearization of the plasmid with appropriate
restriction enzymes. The riboprobes were labeled using a
digoxigenin-labeling kit (Boehringer-Mannheim, Indianapolis, IN).
Statistics. Each uptake experiment was done in triplicate and repeated two or three times. Uptake values given are means ± SE of these replicates. Statistical analysis was done using Student's t-test, and P < 0.05 was considered significant.
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RESULTS |
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Structural features of mouse OCT3.
The mouse kidney OCT3 cDNA (GenBank accession number AJ001417) is
3,391-bp long with an open reading frame of 1,656 bp (including the
termination codon), encoding a protein of 551 amino acids. The
predicted molecular mass of the protein is 61 kDa. Hydrophobicity
analysis using the algorithm of Kyte and Doolittle (13)
and with 20-21 amino acid residues per membrane-spanning domain
indicates that the protein possesses 12 putative transmembrane domains.
There are four potential sites for N-linked glycosylation at positions
72, 99, 114, and 199 in putative extracellular domains. A comparison of
the amino acid sequence of mouse OCT3 with rat OCT3 reveals that they
have 98% identity. In addition, the mouse OCT3 amino acid sequence
bears significant homology to the other members of the organic cation
transporter gene family (Table 1). At the
level of amino acid sequence, mouse OCT3 has 47-50% identity and
66-68% similarity with mouse OCT1 and mouse OCT2. In addition, the amino acid sequence of this clone is identical to that of the
organic cation transporter recently cloned from mouse placenta (26).
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Functional expression of mouse OCT3 in mammalian cells.
Even though Verhaagh et al. (26) have recently reported
the cloning of mouse OCT3 from placenta that is identical to the mouse
OCT3 cloned from kidney in the present study, there is no information
available on the functional characteristics of the mouse OCT3.
Therefore, we expressed the mouse kidney OCT3 heterologously in HRPE
cells using the vaccinia virus expression technique. TEA was used as
the prototypical organic cation to assess the transport function of
OCT3 (Fig. 1). Expression of mouse OCT3
in HRPE cells induced the uptake of TEA severalfold compared with
vector-transfected cells. The mouse OCT3-mediated TEA uptake was pH
dependent (Fig. 1A). The uptake was enhanced approximately
threefold when the pH of the extracellular medium was changed from 5.5 to 8.5. The substrate specificity of mouse OCT3 was investigated by
assessing the effect of various organic cations on the mouse
OCT3-mediated uptake of [14C]TEA in vector-transfected
and mouse OCT3 cDNA-transfected cells under identical conditions.
Figure 1B describes the results for mouse OCT3-specific
uptake (i.e., uptake in cDNA-transfected cells minus uptake in
vector-transfected cells). The mouse OCT3-mediated uptake of
[14C]TEA was inhibited by all organic cations tested.
When the concentration of [14C]TEA was 20 µM, the
inhibition caused by various organic cations at a concentration of 5 mM
was in the range of 60-100%. The most potent inhibitors were
dimethylamiloride, MPP,
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), clonidine, and
cimetidine with inhibition >90%. Moderate inhibition (60-70%)
was observed with N1-methylnicotinamide and
guanidine. The mouse OCT3-specific TEA uptake was saturable
over a TEA concentration of 0.5-15 mM, and the Michaelis-Menten
constant for the process was 1.9 ± 0.2 mM.
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Functional expression of mouse OCT3 in X. laevis oocytes.
The mouse OCT3 was also expressed in X. laevis oocytes by
microinjection of mouse OCT3 cRNA into the oocytes. Water-injected oocytes served as control. As shown in Fig.
2A, the uptake of TEA,
guanidine, and MPP was severalfold higher in cRNA-injected oocytes
compared with uptake in water-injected oocytes.
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Structural organization of the mouse OCT3 gene.
The BAC clone containing the full-length mouse OCT3 gene was
analyzed by a combination of techniques that included restriction enzyme digestion, Southern hybridization with mouse OCT3 cDNA, subcloning and sequencing of the hybridization-positive fragments, and
PCR to determine the sequence of the linking regions between the
fragments. Using this approach, we could determine the sequence of the
mouse OCT3 gene in its entirety except for the first and seventh introns. These two introns could not be amplified by PCR, and
therefore the size and sequence of these introns could not be
determined. A comparison of the nucleotide sequences of the gene and
cDNA has enabled us to deduce the exon-intron organization of the gene
(Fig. 3). The gene consists of 11 exons
and 10 introns. Exon 1 contains the 5'-untranslated region and a part
of the coding region. The 3'-untranslated region is present in exon
11. All exon-intron boundaries conform to the consensus
donor/acceptor sequence (gt/ag) for RNA splicing. The details of the
exons and introns are given in Table 2.
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Regional distribution of OCT3 mRNA in the kidney.
We analyzed the distribution pattern of OCT3 mRNA in mouse kidney by in
situ hybridization (Fig. 4). Analysis of
sagittal sections of normal mouse kidney with antisense riboprobe
revealed abundant expression of OCT3 mRNA in the cortical region with
little or no expression in the medulla. The signals were specific as evidenced from the absence of hybridization with sense riboprobe. Higher magnification of the cortical region revealed the expression of
OCT3 mRNA throughout the cortex with notable exception of renal corpuscles. The signal was seen specifically in proximal and distal convoluted tubules and within Bowman's capsule but was absent in the
glomerulus.
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Expression of OCT3 in human kidney.
We cloned OCT3 originally from rat placenta (10). Northern
blot analysis has shown that, among various rat tissues tested, OCT3
mRNA is expressed most prominently in the placenta and moderately in
the intestine and heart. Low but detectable levels of mRNA are present
in the brain and kidney. We have now cloned human OCT3 from placenta
and HKPT cell line. The amino acid sequence of human OCT3 is identical
to the human EMT recently cloned by Grundemann et al. (6)
from Caki-1 cells, a human kidney carcinoma cell line. At the level of
amino acid sequence, human OCT3 exhibits 86% identity and 90%
similarity to mouse OCT3. In addition, human OCT3 shows significant
homology to other members of the organic cation transporter family
(Table 1). We then used the human OCT3 cDNA as a probe to examine the
distribution of OCT3 mRNA in human tissues by Northern blot analysis
(Fig. 5). The OCT3-specific hybridization
signal was strong in several tissues including the liver, placenta,
kidney, and skeletal muscle. The signal was comparatively weak but
readily detectable in the lung, heart, and brain. This expression
pattern of OCT3 in human tissues agrees with the results reported
recently by Verhaagh et al. (26). We also investigated the
expression of OCT3 mRNA in several human cell lines. The OCT3-specific hybridization signal was detectable only in a cervical carcinoma cell
line (HeLa) and a kidney proximal tubular cell line (HKPT). The signal
was not detectable in two placental trophoblast cell lines (JAR and
BeWo), two intestinal cell lines (Caco-2 and HT-29), and one breast
cancer cell line (MCF-7) (data not shown).
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Functional expression of human OCT3 cDNA in HRPE cells.
To establish the functional identity of human OCT3 as an organic cation
transporter, we used the vaccinia virus expression system to express
human OCT3 cDNA in HRPE cells. Cells transfected with vector alone
served as control. We studied the uptake of three organic cations (TEA,
MPP, and guanidine) in these cells (Fig.
7). The uptake of TEA and MPP was
severalfold higher in cDNA-transfected cells than in vector-transfected
control cells. The cDNA-specific guanidine uptake was comparatively
low, but the uptake in cDNA-transfected cells was significantly higher than in control cells (P < 0.05). We also analyzed the
kinetics of MPP uptake mediated by human OCT3 (Fig.
8A). The uptake was saturable
with a Michaelis-Menten constant of 47 ± 5 µM. We further analyzed the substrate specificity of human OCT3 by assessing the
ability of various organic cations to inhibit the uptake of [3H]MPP mediated by human OCT3 (Fig. 8B).
Seven different organic cations were examined, and all of them were
found to inhibit human OCT3-mediated [3H]MPP uptake. The
relative potency of inhibition was in the following order:
desipramine > imipramine > MPP > clonidine > procainamide > TEA > guanidine. The corresponding
inhibition constants (Ki) were 14 ± 3, 42 ± 16, 54 ± 2, 373 ± 78, 738 ± 198, 1,372 ± 278, and 6,201 ± 69 µM, respectively.
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DISCUSSION |
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OCT3 is an organic cation transporter originally cloned from rat
placenta (10). It handles a variety of cationic drugs in addition to the endogenous organic cations such as dopamine,
norepinephrine, and histamine. Subsequent studies (27)
have revealed that OCT3 is identical to the EMT (uptake2)
that has been described functionally as a transporter specific for
monoamines (24). The functional characteristics of the EMT
include the transport of dopamine and norepinephrine with low affinity,
independence of the presence of Na+ or Cl,
and sensitivity to inhibition by steroids (24). This
transporter was named "uptake2" to differentiate it
from uptake1, which is a Na+- and
Cl
-coupled transporter specific for the monoamine
norepinephrine expressed in noradrenergic neurons (17).
OCT3 exhibits all of these characteristics. Recent studies by
Grundemann et al. (6) with human OCT3 have confirmed the
identity of OCT3 as the EMT.
The present study was undertaken to examine the expression of OCT3 in the kidney. The expression of OCT1 and OCT2, the first two organic cation transporters to be cloned, has been studied in the kidney, but there is no information available regarding the expression of OCT3 in this tissue. Since OCT3 is capable of transporting various organic cations, the relevance of this issue to the physiological function of the kidney is readily apparent. The renal tubular cells play an important role in the excretion/absorption of cationic xenobiotics. One of the steps involved in the renal excretion of cationic drugs is the entry of these drugs from the blood into the cells across the basolateral membrane via a potential-sensitive transport mechanism. Two different potential-sensitive organic cation transporters, namely OCT1 and OCT2, have been shown to be expressed in the kidney (12). OCT1 is expressed in the rodent kidney but not in the human kidney, whereas OCT2 is expressed in the rodent as well as human kidneys (12). The present studies provide evidence for the expression of OCT3 in the kidney. The data show that this transporter is expressed in the rodent kidney as well as in the human kidney. The expression of OCT3 mRNA is specific to the proximal and distal convoluted tubules in the mouse kidney. The fact that OCT3 is also capable of transporting the monoamines dopamine, norepinephrine, and histamine implies that this transporter may also influence the renal disposition of these endogenous compounds.
The present study also describes the genomic organization of the mouse OCT3 gene. This is the first report on the genomic organization of a potential-sensitive organic cation transporter. There is no information available on the structure of the genes encoding OCT1 and OCT2. It is, however, interesting to note that the genes for all three known potential-sensitive organic cation transporters, namely OCT1, OCT2, and OCT3, are located very close to each other on the same chromosome 17 in mouse (26). The same is true in humans. The genes for OCT1, OCT2, and OCT3 are located very close to each other on chromosome 6 in humans (6, 12). This suggests that all three genes may have evolved from a common ancestor gene.
Our results with the functional characteristics of human OCT3 are significantly different from those of Grundemann et al. (7). The recent report by these investigators has claimed that human OCT3 transports MPP but not TEA. Our present studies, however, show convincingly the ability of human OCT3 to transport TEA as well as MPP. In fact, the relative increase in uptake in human OCT3 cDNA-transfected cells compared with control cells is similar for TEA and MPP. The claim by Grundemann et al. (7) that human OCT3 is specific for endogenous monoamines and does not accept cationic xenobiotics is not supported by our current study. These investigators have also concluded that human OCT3 does not have a broad substrate specificity and is therefore not polyspecific. Our current studies do not agree with this conclusion either. We have shown in the present study that human OCT3 interacts with not only TEA but also with a variety of cationic xenobiotics such as MPP, clonidine, imipramine, desipramine, and procainamide. Finally, Grundemann et al. (7) claim that human OCT3 corresponds to the guanidine/H+ antiport mechanism that we have described previously in human placental (3) and rabbit intestinal (14) brush-border membrane vesicles. However, there is no evidence to support this conclusion. Our studies with brush-border membrane vesicles have shown that the transporter responsible for guanidine/H+ antiport does not interact with TEA (3, 14). In contrast, human OCT3 interacts very well with TEA and shows comparatively very little affinity toward guanidine. Among various organic cations tested, guanidine has the lowest affinity for human OCT3. Furthermore, the present studies show clearly that the transport of TEA and MPP via human OCT3 is electrogenic. This would not be expected if human OCT3 operates as an organic cation/H+ antiporter. Therefore, we conclude that human OCT3 is a potential-sensitive polyspecific organic cation transporter similar to rat OCT3 and mouse OCT3.
OCT1 and OCT2 have been shown to be present in the basolateral membrane of renal tubular epithelial cells where they mediate the membrane potential-dependent entry of organic cations from the blood into the cells (19). We speculate that OCT3 is also present in the basolateral membrane mediating the potential-dependent entry of organic cations. OCT3-specific antibodies are needed, however, to support this speculation. Human kidney expresses only OCT2 and OCT3. The expression of OCT2 in human kidney is restricted to distal convoluted tubules (4). Since renal secretion of organic cations occurs primarily in the proximal tubule (19, 25), OCT2 may not be the primary transporter involved in this process. On the other hand, OCT3 is expressed abundantly in the proximal tubule as evidenced by in situ hybridization data in the mouse kidney. Similar localization studies with OCT3 in the human kidney have not been done. If the expression pattern of OCT3 in the human kidney is similar to that in the mouse kidney, then OCT3 is likely to play an important role in the renal secretion of organic cations in humans.
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ACKNOWLEDGEMENTS |
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We thank Vickie Mitchell for excellent secretarial assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grant DA-10045.
Address for reprint requests and other correspondence: V. Ganapathy, Dept. of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100 (E-mail: vganapat{at}mail.mcg.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 15 November 1999; accepted in final form 9 May 2000.
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