Molecular cloning of rabbit organic cation
transporter rbOCT2 and functional comparisons with
rbOCT1
Xiaohong
Zhang,
Kristen K.
Evans, and
Stephen H.
Wright
Department of Physiology, College of Medicine, University
of Arizona, Tucson, Arizona 85724
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ABSTRACT |
Multiple organic cation
transporters (OCTs) are present in rabbit kidney and may play different
functional roles. We cloned rabbit OCT2 (rbOCT2) and compared its
function with that of rabbit OCT1 (rbOCT1). In transiently transfected
COS-7 cells, rbOCT1 and rbOCT2 mediated uptake of
[3H]tetraethylammonium (TEA) with
Kt values of 188 and 125 µM, respectively. n-Tetraalkylammonium compounds showed similar affinities for
the two homologs, with IC50 values for inhibition of OCT1-
and OCT2-mediated [3H]TEA transport, respectively, of
4,538 and 1,395 µM for tetramethylammonium, 88.5 and 3.9 µM for
tetrapropylammonium, 13.9 and 5.3 µM for tetrabutylammonium, and 8.8 and 7.6 µM for tetrapentylammonium. However, the transporters had
very different affinities for cimetidine (CIM): IC50 of 916 and 5.7 µM for rbOCT1 and rbOCT2, respectively. CIM inhibition of TEA
uptake into single S2 segments of rabbit proximal tubule was used to
estimate the contributions of OCT1 and OCT2 to basolateral organic
cation uptake. The median IC50 for CIM inhibition of TEA uptake was 12.3 µM, suggesting that OCT2 is the major contributor to
basolateral organic cation transport in the S2 segment of proximal tubule in rabbit kidney.
kidney; proximal tubule; uptake; tetraethylammonium; rabbit organic
cation transporter
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INTRODUCTION |
THE RENAL TUBULAR
TRANSPORT of organic substances plays an essential role in the
clearance from the body of xenobiotics, such as drugs, numerous
chemicals in our environment, and some metabolites. In general, the
sequence of tubular secretion of organic cations involves basolateral
uptake, accumulation into the cell, and subsequent extrusion from the
cell into tubular fluid across the luminal membrane of renal epithelial
cells (35). Secretion and reabsorption of organic cations
have been described in renal proximal tubules but may also occur in
distal tubules or collecting ducts (1, 2, 6, 24). The
mechanisms mediating tubular secretion have been intensively
studied in various experimental preparations from rabbit kidney,
including perfused (9, 24, 38) and nonperfused (5,
10, 15, 38) tubules, apical (12, 26, 29, 44, 45)
and basolateral (28, 39, 44) membrane vesicles, and
isolated tissue slices (21). Results from these studies
identified two distinct functional classes of organic cation
transporter (OCT): one localized at the basolateral membrane that is
stimulated by the inside-negative membrane potential of proximal cells
and the other at the brush-border membrane that is stimulated by an
inwardly directed proton gradient (35).
Expression and molecular cloning strategies have been used to identify
at least five OCT members from mammalian tissues, including OCT1, OCT2,
OCT3, OCTN1, and OCTN2 (8). Among these members, apparent
expression levels implicate OCT1 and OCT2 as major renal organic
transporters that use membrane potential as a driving force. OCT1 has
been cloned in the rat (17), human (46),
mouse (14), and rabbit (42). OCT2, in
addition to the rat, mouse, and human (13, 30, 32), has
been cloned from the pig (16); OCT2 has not been cloned
from the rabbit. There are distinct species differences in the tissue
distribution of OCT1 and OCT2. Rat OCT1 (rOCT1) is expressed most
abundantly in the kidney, moderately in the liver, and at very low
levels in the intestine (17). In contrast, human OCT1 is
expressed abundantly in the liver, is scarce in the kidney, and is not
evident in the intestine (13, 46). In the rabbit, OCT1
(rbOCT1) distribution is quantitatively similar to that in the human,
with highest expression in the liver, although mRNA transcripts are
detectable at significant levels in the kidney and intestine
(42). In the human, rat, and mouse, OCT2 expression is
largely confined to the kidney (13, 30, 32).
Immunocytochemistry of cortical tissue from the rat (20, 41) and human (31) confirms that OCT1 and OCT2
expression is restricted to the basolateral membrane of proximal tubule
cells. In the rat, OCT1 expression is largely restricted to the early (S1) and middle (S2) segments of the proximal tubule, with OCT2 expression restricted to the S2 and S3 segments (20).
Thus, in the rat, OCT1 and OCT2 are coexpressed in (at least) the S2 segment of the proximal tubule. In the human, only OCT2 expression is
evident in proximal cells (31). Importantly, neither
immunolocalization nor in situ hybridization provides evidence on the
functional distribution of transport activity of coexpressed
transporter homologs. Functional differences in expression of OCT
homologs are not understood in any species.
The rabbit kidney offers one of the few working models of intact renal
tubule function, so it is of considerable interest to understand the
physiological roles of OCT1 and OCT2 within the proximal tubule. To
this end, we have cloned rabbit OCT2 (rbOCT2) and compared functional
characteristics and subsegmental distribution of rbOCT1 and rbOCT2 in
the proximal tubule. Although the two homologs had similar substrate
specificities, rbOCT2 generally showed a higher affinity for substrates
than rbOCT1. Subsegmental localization studies by PCR suggested that
only rbOCT2 was expressed in S2 segments in the rabbit kidney. On the
basis of the profile of cimetidine inhibition of tetraethylammonium
(TEA) transport in cells expressing OCT1 or OCT2 and in isolated single
S2 segments of the proximal tubule, we concluded that OCT2 is the major
contributor to basolateral organic cation transport in the S2 segment
of the proximal tubule in the rabbit kidney.
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METHODS |
Materials.
[3H]TEA (13 Ci/mmol) was acquired from American
Radiolabeled Chemicals (St. Louis, MO). The fluorescent organic
cation 2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl trimethylammonium (NBD-TMA) was synthesized as described elsewhere (4). All other chemicals were purchased from Sigma
Chemical (St. Louis, MO). The monkey kidney cell line COS-7 was
obtained from the American Type Culture Collection (Rockville, MD). The mammalian expression vector pcDNA3.1 was purchased from Invitrogen (Carlsbad, CA). Cell culture media and all other molecular biology reagents were purchased from Life Technologies (Gaithersburg, MD). New
Zealand White rabbits were purchased from Myrtle's Rabbitry (Thompson
Station, TN).
Isolation of mRNA.
Total RNA was prepared from rabbit kidney tissues or isolated rabbit
kidney tubules following the method of Sambrook et al. (37). Organs were removed from anesthetized animals and
extensively washed or perfused with saline buffer to remove most of the
remaining blood or further dissected to prepare renal tubules.
Poly(A)+ RNA was selected on oligo(dT) cellulose
columns from the total RNA preparation and analyzed by agarose gel electrophoresis.
Isolation of rbOCT1 and rbOCT2.
The open reading frame of rbOCT1 was amplified with primers on the
basis of published sequences (42) using Pfu DNA
polymerase and then subcloned into pcDNA3.1 vector. To clone rbOCT2,
degenerate sense and antisense oligonucleotide primers were designed
from consensus sequences of human, rat, mouse, and pig OCT2 as follows: 5'-GTCAGAACYCCTCAGATAAG-3'(sense) and
5'-GATGCCRCCRATGTCAC-3'(antisense). For first-strand synthesis,
0.5 µg of rabbit kidney poly(A)+ RNA was reverse
transcribed using Moloney murine leukemia virus reverse transcriptase
(RT) H
at 37°C for 20 min. After incubation at 70°C
for 15 min, ribonuclease (RNase) H was added, and the reactions were
kept again at 37°C for 20 min. The RT reaction (2 µl) was used
directly for amplification. The PCR solution was assembled and heated
at 94°C for 3 min before Pfu DNA polymerase was added.
Subsequently, PCR was performed using the following profile: 94°C for
1 min, 54°C for 1 min, and 72°C for 2 min for 35 cycles. The last
cycle was terminated after an elongation time of 7 min. A 406-bp RT-PCR
product was gel purified and sequenced. To obtain the remaining 5' and
3' portions of the rabbit kidney OCT2 sequence, the PCR-based 5'- and
3'-rapid amplification of cDNA ends (RACE) systems (GIBCO-BRL) were
utilized. Briefly, two gene-specific primers
[5'-GGAAGCACACCTGCATCTTG-3' (sense) and
5'-GAGATTCCTGATGAACGTGG-3' (antisense)] were designed from the
partial rbOCT2 sequence. The 5'- and 3'-RACE reactions were primed with
an internal gene-specific primer and an adapter primer. The PCRs were
performed according to the manufacturer's protocols. The RACE products
were gel purified and subcloned into the mammalian expression vector
pcDNA3.1. The two overlapped RACE products were digested by
BamHI/EcoRI and then ligated to form a
full-length cDNA of rbOCT2. rbOCT1 and rbOCT2 sequences were confirmed
in the sense and antisense strands by an Applied Biosystems model 373A
sequencing unit at the University of Arizona sequencing facility.
Cell culture and transfection.
COS-7 cells were grown at 37°C in a humidified atmosphere (5%
CO2) in plastic culture flasks. The medium was Kaighn's
modification (F12K) medium supplemented with 10% fetal calf serum. The
medium was changed every day, and the culture was split every 3 days. Cells were transfected with supercoiled plasmid DNA by electroporation. Briefly, cells were transfected with 10 µg of DNA at 260 V for 1,050 ms and seeded in 12-well plates at 320,000 cells/well. Uptake studies
were performed 48 h after transfection (cells were generally confluent at this time). Expression of rbOCT1 or rbOCT2 was verified by
RT-PCR and by visual inspection of the accumulation of the fluorescent
cationic dye NBD-TMA (4).
Transport assays.
Uptake was measured at 25°C. After a preincubation period of 30 min
with Waymouth's buffer (WB; in mM: 135 NaCl, 13 HEPES-NaOH, pH 7.4, 28 D-glucose, 5 KCl, 1.2 MgCl2, 2.5 CaCl2, and 0.8 MgSO4), the cells were incubated
with ~76 nM 3H-labeled substrates in WB. Incubation was
stopped by rinsing the cells three times with 2 ml of ice-cold WB
containing 250 µM tetrapentylammonium (TPeA). The cells were then
solubilized with 0.2 N NaOH and 1% (vol/vol) SDS in 1 N HCl, pH 7.4, and radioactivity was determined by liquid scintillation spectrometry.
Uptakes are expressed as moles per square centimeter of nominal cell
surface of the confluent monolayer.
Transport in isolated tubules.
New Zealand White rabbits were killed by intravenous injection of
pentobarbital sodium. The kidneys were flushed via the renal artery
with an ice-chilled solution containing 250 mM sucrose and 10 mM HEPES
adjusted to pH 7.4 with Tris base. The kidneys were removed and sliced
transversely, and the slices were placed in a dish containing
ice-chilled dissection buffer (in mM: 110 NaCl, 25 NaHCO3,
5 KCl, 2 Na2HPO4, 1.8 CaCl2, 1 MgSO4, 10 sodium acetate, 8.3 D-glucose, 5 L-alanine, 4 lactate, and 0.9 glycine) adjusted to pH 7.4 with HCl or NaOH and gassed continuously with 95% O2-5%
CO2 to maintain the pH (osmolarity ~290
mosmol/kgH2O). Dissection of tubules from a slice was
performed manually at 4°C without the aid of enzymatic agents.
Dissections were limited to isolation of early proximal straight
tubules (S2 segments), defined as extending from the cortical surface
to the corticomedullary junction (38). Uptake of
[3H]TEA into tubules was started by transfer of
individual tubule segments to a chamber containing 37°C uptake medium
with labeled substrate and, in some cases, unlabeled cimetidine. Uptake
was terminated by individual transfer of the tubule segments to 10 µl
of 6 N NaOH that was dispensed into microwells of a plastic 60-well
plate (Nunc, Naperville, IL). A 10-µl syringe was used to transfer
the NaOH solution (and the tubule segment) to separate plastic
scintillation vials that contained 300 µl of distilled water. Each
microwell was rinsed twice with 10 µl of distilled water that was
added to its respective scintillation vials. The radioactivity in each
vial was measured using liquid scintillation spectrometry. Three to
four tubule segments were used for each experimental and each control condition.
RT-PCR analysis.
RT-PCR was performed with mRNA from isolated S2 segments and rbOCT1-
and rbOCT2-specific primers: for rbOCT1 a 499-bp fragment derived from
5'-ATGGTGTGTTCTGCGCTA-3' (sense) and 5'-CCACTGGAACAGGAAGCA-3' (antisense) and for rbOCT2 a 406-bp fragment derived from
5'-GTCAGAACYCCTCAGATAAG-3' (sense) and 5'-GATGCCRCCRATGTCAC-3'
(antisense). Subsequent PCR was carried out in separate reactions
employing identical parameters using primers for rbOCT1 or rbOCT2 and
equivalent amounts of tubule RT reaction.
Data analysis.
Uptake values are presented as means ± SE. In each experiment, a
minimum of three wells was used to generate each data point, and each
experiment was repeated at least three times.
Amino acid sequences and pairwise sequence alignments were analyzed
with default parameters with the ClustalW algorithm available on the
internet from Network Protein Sequence Analysis
(http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_server.html).
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RESULTS |
Molecular properties of rbOCT2.
A 406-bp RT-PCR product was generated by PCR amplification using
degenerate oligonucleotide primers designed from consensus sequences of
human, rat, mouse, and pig OCT2. PCR-based 5'- and 3'-RACE systems were
utilized to obtain the remaining 5' and 3' portions of the rbOCT2
sequence. The two overlapped RACE products were digested by
BamHI/EcoRI and then subcloned into the mammalian expression vector pcDNA3.1 to form a full-length cDNA of rbOCT2. The
nucleotide and predicted amino acid sequences of rbOCT2 are shown in
Fig. 1. The full-length cDNA is 2,180 bp
and contains a 42-bp 5'-untranslated region, a 1,662-bp open reading
frame, and a 476-bp 3'-untranslated region. It encodes a protein of 554 amino acids with a predicted mass of 61 kDa (GenBank accession no.
AF458095). Assessment of possible secondary structure (TMHMM, version
2.0) (27, 40) suggests the presence of two large
hydrophilic loops and 12 membrane-spanning domains, which is similar to
that of rbOCT1 and other OCT2 isoforms (8). The protein
sequence contains five potential N-linked glycosylation sites (N-X-T/S) at positions 71, 96, 112, 198, and 331, with the
first two sites (positions 71 and 96) conserved among OCT1,
OCT2, and OCT3 (8). In addition, one potential protein
kinase A phosphorylation site (position 344), two potential
tyrosine kinase phosphorylation sites, and three potential protein
kinase C (PKC) phosphorylation sites (positions 59, 285, and
319) were identified (Fig. 1).

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Fig. 1.
Nucleotide (bottom rows) and predicted amino acid
(top rows) sequence of the rabbit organic cation transporter
cDNA rbOCT2. Nucleotides are numbered at left; amino acids
are numbered at right. , putative N-glycosylation site;
, consensus protein kinase A site; ,
consensus protein kinase C site; *, stop codon at nucleotide 1705 (TAA).
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BLAST searches of the protein and gene databases indicated that the
rbOCT2 protein belongs to the OCT family of the Major Facilitator
Superfamily (2.A.1.19) (36). rbOCT2 has high sequence homology with its orthologs cloned from the human, rat, mouse, and pig
(82-84% identity) and strong homology with the related proteins
OCT1 (68-69% for human, rat, and mouse and 71% for rabbit) and
OCT3 (48-49%). Alignment of the sequences of rbOCT1 and rbOCT2 is
shown in Fig. 2.

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Fig. 2.
Sequence alignment of rbOCT1 and rbOCT2. Amino acid sequences of
rabbit OCT1 (rbOCT1; accession no. AF196774, GenBank) and rbOCT2
(accession no. AF458095, GenBank) were aligned. Amino acids that are
fully conserved are highlighted. Multiple sequence alignment was made
using the Network Protein Sequence Analysis program.
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Functional comparison of rbOCT2 with rbOCT1.
Transient transfection of COS-7 cells with pcDNA3.1-containing OCT2
increased the 5-min uptake of [3H]TEA by 14-fold, and
this was reduced to a level comparable to that observed in the
vector-only control by addition of 2.5 mM unlabeled TEA (Fig.
3). Figure
4 shows the time courses of
[3H]TEA uptake into COS-7 cells transfected with rbOCT1
(Fig. 4A) or rbOCT2 (Fig. 4B). For both
processes, uptake was time dependent and reasonably linear for 2-5
min. Extrapolation of the time courses to time 0 consistently revealed positive intercepts, suggesting that accumulation
of labeled TEA might include a rapid binding component. However, the
rapid component of cellular TEA accumulation was completely blocked by
addition of unlabeled TEA with kinetics that were indistinguishable
from those of the time-dependent portion of uptake (data not shown). In
addition, a rapid component of accumulation was absent in experiments
with wild-type COS-7 cells and cells transfected with empty vector
(data not shown), indicating that it reflected expression of transport
protein. Therefore, in subsequent kinetic studies, 2- and 5-min uptakes
were used as estimates of the initial rates of transport mediated by
OCT1 and OCT2, respectively.

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Fig. 3.
Tetraethylammonium (TEA) uptake in COS-7 cells
transfected with pcDNA3.1 (vector) or pcDNA3.1/rbOCT2 was measured at
room temperature with 76 nM [3H]TEA 48 h after
transfection. Unlabeled TEA (2.5 mM) was added for uptake in the
inhibition experiment. Each point represents mean ± SE of
substrate accumulation in 3 individual experiments with 3 wells of
cells for each experiment.
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Fig. 4.
Time course of rbOCT1- and rbOCT2-mediated transport of
[3H]TEA in COS-7 cells. Uptake of [3H]TEA
was measured 48 h after transfection. pcDNA3.1-rbOCT1
(A) and pcDNA3.1-rbOCT2 (B) cells were incubated
for specified time periods at room temperature in buffer containing 76 nM [3H]TEA. After incubation, radioactivity
[disintegrations per minute (DPM)] of solubilized cells was counted.
Each point represents mean ± SE of 3 individual experiments with
3 wells of cells for each experiment.
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Next, we examined the concentration dependence of [3H]TEA
uptake by rbOCT1 and rbOCT2. The transfectants were exposed to
[3H]TEA and increasing concentrations of unlabeled TEA
(0-2.5 mM). Inhibition of labeled TEA uptake produced by unlabeled
TEA was used to calculate the kinetics of TEA transport according to
the following relationship (23)
where J is the rate of [3H]TEA transport
from a concentration of labeled substrate equal to [*T],
Jmax is the maximum rate of mediated TEA
transport, Kt is the TEA concentration that
results in half-maximal transport (Michaelis constant), [T] is the
concentration of unlabeled TEA in the transport reaction, and
C is a constant that represents the component of total TEA
uptake that is not saturated (over the range of substrate
concentrations tested) and presumably reflects the combined influence
of diffusive flux, nonspecific binding, and/or incomplete rinsing of
the cell layer. For OCT1 and OCT2, addition of unlabeled TEA resulted
in a hyperbolic inhibition of labeled TEA uptake with <10% of
accumulated label in the "nonsaturable" component. Figure
5 shows the kinetics of rbOCT1- and
rbOCT2-mediated TEA transport in representative experiments. In three
separate experiments on cells expressing rbOCT1,
Jmax was 49.7 ± 2.73 pmol · cm
2 · min
1 with a
Kt of 188 ± 20 µM. For rbOCT2,
Jmax was 13.1 ± 0.16 pmol · cm
2 · min
1 with a
Kt of 125 ± 22 µM.

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Fig. 5.
Comparison of kinetics of TEA transport in transient
transfected COS-7 cells expressing rbOCT1 (A) or rbOCT2
(B). Uptakes of [3H]TEA at 2 and 5 min were
measured for rbOCT1 and rbOCT2, respectively. Transport buffer
contained increasing concentrations of unlabeled TEA (0-2.5 mM).
For representative experiments shown, the Michaelis constant
(Kt) for TEA transport mediated by rbOCT1 was
189 µM, with a maximum velocity (Jmax) of 47.9 pmol · cm 2 · min 1.
Inset: total velocity (Jtotal)-total
TEA concentration plot of these data using a nonlinear fit to a model,
consisting of one saturable (Michaelis-Menten) term plus one
nonsaturable term, the analysis of which resulted in
Kt of 168 µM with Jmax
of 44.3 pmol · cm 2 · min 1.
For rbOCT2, Kt was 106 µM with
Jmax of 12.8 pmol · cm 2 · min 1
(inset: Kt = 122 µM with
Jmax = 13.1 pmol · cm 2 · min 1). Data
points are means ± SE of uptakes measured in 3 wells from single
representative transfections.
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To examine the characteristics of substrate recognition,
accumulation of [3H]TEA by rbOCT1- and rbOCT2-expressing
cells was measured in the presence of increasing concentrations of
several organic cations. All the test agents inhibited TEA uptake by
rbOCT1 and rbOCT2 cells (Table 1),
suggesting that both transporters recognize a wide variety of cationic
molecules. OCT2 displayed a higher apparent affinity for every compound
tested. These differences are most evident by considering the ratio of
IC50 values {inhibitor concentrations that reduced
mediated uptake of [3H]TEA by 50%, determined using the
analytic method of Groves et al. (15)} obtained for
rbOCT1 relative to that obtained for rbOCT2 (Fig.
6). In some cases, the ratio was
comparatively small, e.g., ratios of 1.2 and 1.5 for TPeA and TEA,
respectively, indicating that the two homologs had similar affinities
for these molecules. However, several compounds showed substantially
higher affinities for rbOCT2 than for rbOCT1. Cimetidine showed the
most marked difference in apparent affinity for the two transporters
(Figs. 6 and 7), with IC50
160 times higher for rbOCT1 than for rbOCT2 (916 vs. 5.7 µM; Table
1). The fluorescent cation NBD-TMA also displayed a much higher
affinity for rbOCT2 (IC50 = 3.6 µM) than for rbOCT1
(IC50 = 129 µM; Table 1, Fig. 6).
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Table 1.
Inhibition constants for organic cations to compete with
[3H]TEA uptake mediated by rbOCT1- and rbOCT2-transfected
COS-7 cells
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Fig. 6.
Ratio of IC50 values obtained for a
battery of inhibitors of TEA uptake into COS-7 cells. To calculate
ratios, values obtained for rbOCT1 and rbOCT2 were "numerator" and
"denominator," respectively. Absolute IC50 values are
listed in Table 1. TPeA, tetrapentylammonium; TBA, tetrabutylammonium;
TMA, tetramethylammonium; TPrA, tetrapropylammonium; MPP,
1-methyl-4-phenylpyridinium; NBD-TMA,
2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl trimethylammonium.
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Fig. 7.
Effects of cimetidine on TEA uptake by rbOCT1 and rbOCT2.
COS-7 cells transfected with rbOCT1 (A) or rbOCT2
(B) were incubated for 2 or 5 min, respectively, at room
temperature with 76 nM [3H]TEA in the presence of
0-500 µM cimetidine. After incubation, radioactivity of
solubilized cells was counted. Data points are means ± SE of
uptakes measured in 3 wells from single representative transfections.
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Distribution of rbOCT1 and rbOCT2 in the S2 segment of the rabbit
proximal tubule.
The marked difference in apparent affinity of cimetidine for the two
rabbit OCT homologs suggested that cimetidine could be used as a tool
to examine the relative contribution of OCT1 and OCT2 to basolateral
organic cation transport in the intact proximal tubule. Figure
8 shows the effect of increasing
concentrations of cimetidine on the basolateral uptake of
[3H]TEA into single, nonperfused S2 segments of rabbit
renal proximal tubule. Cimetidine proved to be a high-affinity
inhibitor of basolateral TEA uptake. In five separate experiments with
tubules from different rabbits, the average IC50 for
cimetidine's inhibition of TEA uptake was 19.5 ± 8.4 µM. In
one of those experiments, the IC50 was particularly high
(52 µM), and we consider the median value of 12.3 µM to be more
representative of the inhibitory effect of cimetidine on basolateral
organic cation transport in intact S2 segments. This value is very
similar to 10 µM, which was reported to produce half-maximal
steady-state accumulation of cimetidine in single rabbit S2 segments
(7). With respect to the issue of which OCT homolog is
expressed in proximal S2 segments, the IC50 of ~12 µM
in the intact tubule was more comparable to the IC50 of 6 µM against rbOCT2 than to ~900 µM for inhibition of rbOCT1. This
supports the conclusion that OCT2 is the major contributor to
basolateral organic cation transport (at least for TEA and cimetidine)
in the S2 segment of the proximal tubule in rabbit kidney.

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Fig. 8.
Effects of cimetidine on TEA uptake by S2 segment of
rabbit renal proximal tubule. Nonperfused S2 segment was incubated for
5 min at 37°C with 1.8 µM [3H]TEA in the presence of
0-1 mM cimetidine. After incubation, radioactivity of solubilized
tissue was counted. Each data point is mean uptake measured in 3-4
tubule segments determined in 5 separate animals.
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We also compared mRNA expression of rbOCT1 and rbOCT2 in individual S2
segments of rabbit renal tubule by RT-PCR. In control experiments, the
primers were shown to amplify appropriately sized products using the
respective cDNAs as templates (data not shown). However, when the RT
material derived from single S2 segments (single tubules from 3 rabbits) was used as the template, amplified product was only ap-parent
using the primers for OCT2 (Fig. 9), consistent with the conclusion that OCT2 mRNA is expressed to a much
greater extent in the S2 segment of rabbit proximal tubule than is mRNA
for OCT1.

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Fig. 9.
Detection of rbOCT1 and rbOCT2 mRNA levels by RT-PCR in
single S2 segments of rabbit renal proximal tubule. mRNA prepared from
the tubule segment was used for 1st-strand cDNA synthesis. Subsequent
PCR amplification was performed with rbOCT1- or rbOCT2-specific primers
using the indicated amount of RT reaction. No RT reaction was added to
negative control. PCR products for negative control, rbOCT1, and rbOCT2
were loaded on the same gel and visualized with ethidium bromide.
Lanes 1 and 11, 1-kb DNA ladder; lanes 2, 4, 6, 8, and 10, rbOCT1; lanes 3, 5, 7, and
9, rbOCT2.
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DISCUSSION |
In the kidney, organic cation transport systems play physiological
and pharmacological roles in the excretion and/or reabsorption of a
wide array of endogenous organic cations, cationic drugs, and cationic
toxins. Five members of the OCT family (2.A.1.19) of the Major
Facilitator Superfamily (36) have been cloned in several
mammalian species including human, mouse, rat, rabbit, and pig
(8). However, until the present study, OCT1 was the only
OCT cloned from the rabbit. Much of our understanding of the mechanisms
of renal organic cation transport is based on studies performed in
rabbit kidney, owing to its suitability for the study of the integrated
function of the physiologically intact, isolated perfused tubule. There
are now substantial data suggesting that net renal organic cation
transport could reflect the concerted behavior of separate transport
processes (22). However, substantial species differences
in the quantitative characteristics of organic cation transport,
particularly with respect to the interaction of bulkier organic cations
with orthologous transporters in rodents, rabbits, and humans
(11), make it imprudent to use results obtained with
cloned transporters of one species to predict the transport behavior of
intact tubules in another. Consequently, we cloned the rabbit ortholog
of OCT2 and compared its characteristics with those of its related
renal transport homolog OCT1 to provide new insights into our
understanding of the molecular basis of organic cation transport in
intact renal tubules.
The amino acid identity between rbOCT1 and rbOCT2 is 71%. Computer
analysis reveals 2 large hydrophilic loops and 12 transmembrane-spanning
-helices in rbOCT2, which is similar to
rbOCT1 (and all other members of the OCT family) (8).
There are five potential N-linked glycosylation sites (N-X-T/S) at
positions 71, 96, 112, 198, and 331, with the
first two sites (positions 71 and 96) conserved among OCT1, OCT2, and OCT3. rbOCT2 also possesses three potential PKC
phosphorylation sites at positions 59, 285, and
319, which are located in the two large hydrophilic loops.
Transport studies with isolated rabbit renal proximal tubules
(18) and with the cell lines IHKE-1 and
LLC-PK1 (19) revealed a modulation of organic
cation transport by phorbol esters, and transport activity of rOCT1
(expressed in HEK-293 cells) is increased after activation of PKC
(25). On the other hand, accumulation of the fluorescent cationic dye
4-(4-dimethylaminostyryl)-N-methylpyridinium is
decreased after activation of PKC in isolated human proximal
tubules (34). However, it is not known whether rbOCT2 is
regulated by PKC and which of the potential phosphorylation sites may
be involved in regulation of organic cation transport by PKC.
To compare the functional characteristics of rbOCT1 and rbOCT2, we
transfected rbOCT1 and rbOCT2 into COS-7 cells. The two transporters
had similar apparent affinities for transport of TEA (188 vs. 125 µM,
respectively; Fig. 5), and the ratio of IC50 values
(OCT1/OCT2) was comparatively similar (i.e., ~3-fold difference or
less) for several other organic cations, including the
n-tetraalkylammonium compounds tetrapentylammonium,
tetrabutylammonium, and tetramethylammonium (Fig. 6). However,
substantial differences were noted in the relative affinity of several
organic cations for OCT1 vs. OCT2. The greatest differences were noted
for cimetidine, with an OCT1 IC50 of >900 µM compared
with OCT2 IC50 of 6 µM (Fig. 6, Table 1), and NBD-TMA, with an OCT1 IC50 of 129 vs. 4 µM for OCT2 (Fig. 6, Table
1). These results confirm that in the rabbit, as in other species, OCTs
display a broad specificity for cationic substrates. The results also
indicate that, despite their broad specificity, steric features of
selected substrates must strongly influence binding to the transport
receptors of these closely related homologs.
It is interesting to compare the results obtained here for rbOCT1 and
rbOCT2 with those recently reported for the rat orthologs. It should be
emphasized that such comparisons need to be made cautiously. Here, we
limit the comparisons to those studies that directly compared the
relative affinity of rOCT1 and rOCT2 for a common set of substrates. As
shown in Fig. 10, the similar affinity of rbOCT1 and rbOCT2 for TEA is a characteristic shared by the rat
orthologs of these processes. Nevertheless, it is worth noting that the
absolute values for Kt/inhibition
constant/IC50 values for interaction of TEA with the rat
OCTs varied between studies, ranging from ~100 µM (3)
to 150 µM (33) when expressed in oocytes to ~50 µM
when expressed in Madin-Darby canine kidney (MDCK) cells
(43). The relative interaction of OCT1 and OCT2 with
cimetidine produced the most striking disparities. As noted previously,
in the present study with rbOCTs, there was a 160-fold difference in
the IC50 for cimetidine inhibition of OCT1 (~900 µM)
vs. OCT2 (~6 µM). In two studies comparing the interaction of
cimetidine with rat OCT1 and OCT2, there was virtually no difference in
the relative interaction of OCT1 and OCT2 with this compound. There
was, however, a very large difference in the absolute affinity of the
transporters for cimetidine, with IC50 of ~350 µM when they were expressed in oocytes (33) compared with
IC50 of 6-9 µM when they were expressed in cultured
MDCK cells (43). Similar degrees of variability between
rabbit and rat orthologs and between different systems expressing the
rat homologs were noted for interactions with
1-methyl-4-phenylpyridinium and guanidine. The extent to which these
differences in relative and absolute interactions of organic cations
with OCTs reflect differences in species, expression systems, and/or
technique is not clear. Arndt et al. (3) recently determined the relative interaction of a wide array of substrates for
rOCT1 and rOCT2 (expressed in oocytes) and noted that several compounds
discriminated effectively between these two transporters (notably
mepiperphenidol and O-methylisoprenaline as
OCT2-selective inhibitors and corticosterone as an OCT1-selective
inhibitor). They noted that such marked differences could be used to
dissect out the individual contributions of these processes in intact proximal tubule preparations. However, the variability in interaction of substrates with OCTs noted above, which may reflect species and/or
expression system influences, underscores the importance of using
caution when results obtained with cloned transporters are used to make
predictions about the expected behavior of these processes in native
tissues from other species (e.g., the human).

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|
Fig. 10.
Comparison of relative inhibitory interactions of TEA,
cimetidine (CIM), MPP, and guanidine (Guan) with rabbit or rat
orthologs of OCT1 (numerator) and OCT2 (denominator). Also compared are
these interactions for the 2 rat homologs in 2 separate studies
(3, 33) (cross-hatched and shaded bars, respectively)
employing the oocyte expression system and a third study in which these
transporters were stably expressed in cultured Madin-Darby canine
kidney (MDCK) cells (43). Pairs of numbers in parentheses
above each column are measured Ki/IC50 values
(in µM) for OCT1 and OCT2, respectively.
|
|
Having emphasized this important caveat, it was nevertheless tempting
to use the large difference in apparent affinity for cimetidine of
rbOCT1 and rbOCT2 to examine the level of functional expression of
these two homologs in the S2 segment of rabbit proximal tubule. The
comparatively high affinity of the S2 segment for cimetidine
(IC50 ~ 12 µM; Fig. 8) compared closely with the
high affinity of rbOCT2 (IC50 ~ 6 µM; Fig. 7) and
contrasted markedly with the low affinity of rbOCT1
(IC50 ~ 1 mM; Fig. 7) for this compound. Again, it
is important to acknowledge the underlying assumption that the OCT
affinities expressed in COS-7 cells are those occurring when these
processes are expressed in their native cell type. However, the
similarity between the Kt values for TEA transport observed for OCT1 and OCT2 in COS-7 cells (180 and 120 µM,
respectively) and the Kt for TEA transport in
single proximal tubule segment from rabbit kidney (108 µM)
(15) supports tentative conclusions based on such
comparisons. Moreover, in the present case, RT-PCR showed clear
expression of mRNA for OCT2 in single isolated S2 segments of rabbit
proximal tubules but failed to amplify mRNA for OCT1. Taken together,
these data suggest that OCT function in the S2 segment of proximal
tubule is dominated by OCT2 in the rabbit.
The distribution of OCTs in the rat and human proximal tubule has also
been examined. In the rat, immunocytochemistry (20, 41)
and in situ hybridization (20) indicate that the S2 region of the proximal tubule contains OCT1 and OCT2. The human proximal tubule, in contrast, appears to be dominated by basolateral expression of OCT2 along the entire length of the proximal tubule
(38). Although the failure to find evidence of functional
expression of OCT1 in rabbit S2 segments is consistent with the
expression profile of OCTs in the human tubule, OCT1 is certainly
expressed in rabbit kidney. Northern blots of whole rabbit kidney show
expression of OCT1 mRNA (42). In addition, although RT-PCR
failed to amplify OCT1 in the S2 segments we examined in the present
study, we have seen, on occasion, amplification products in single S2
tubule segments (T. Pannabecker and S. H. Wright, unpublished
observations). Organic cations are secreted along the entire length of
the rabbit proximal tubule, with basolateral accumulation of substrate
being approximately equivalent in the S1, S2, and S3 segments
(38). Thus one or more OCT homologs must be expressed in
the basolateral membrane of each segment. Given the present
observations, it appears that organic cation transport in the mid
portion of rabbit proximal tubule is dominated by activity of OCT2.
In summary, we cloned rbOCT2 and compared its function with that of
rbOCT1 in transiently transfected COS-7 cells. Whereas the two
orthologs have similar Kt values for transport
of [3H]TEA, rbOCT2 generally had a higher affinity for
the battery of organic cations tested. Several substrates, most notably
cimetidine and NBD-TMA, discriminated markedly for rbOCT2 over rbOCT1.
The high-affinity interaction of cimetidine with TEA transport in intact rabbit proximal tubules supported the conclusion that, as in the
human, OCT2 is the major contributor to basolateral organic cation
transport in the S2 segment of the proximal tubule in rabbit kidney.
 |
ACKNOWLEDGEMENTS |
We thank Diane Abbott for preparing the RT material from the S2
segment of the proximal tubule.
 |
FOOTNOTES |
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-58251.
Address for reprint requests and other correspondence:
S. H. Wright, Dept. of Physiology, College of Medicine,
Univ. of Arizona, Tucson, AZ 85724 (E-mail:
shwright{at}u.arziona.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. Section 1734 solely to indicate this fact.
First published January 29, 2002;10.1152/ajprenal.00367.2001
Received 14 December 2001; accepted in final form 15 January 2002.
 |
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