A nucleoside-sensitive organic cation transporter in opossum
kidney cells
Rong
Chen,
Bih Fang
Pan,
Mamoru
Sakurai, and
J. Arly
Nelson
Department of Experimental Pediatrics and Graduate School of
Biomedical Sciences, University of Texas M. D. Anderson Cancer
Center, Houston, Texas 77030
 |
ABSTRACT |
Renal secretion of organic cations and anions
are pleiotropic, active processes in mammals. Some nucleosides such as
deoxyadenosine (dAdo), 2-chlorodeoxyadenosine, and azidothymidine are
secreted by human and rodent kidneys. Previous work (J. A. Nelson, J. F. Kuttesch, Jr., and B. H. Herbert. Biochemical
Pharmacology 32: 2323-2327, 1983) indicated a role
for the classic organic cation transporter (OCT) in the secretion of
the dAdo analog, 2'-deoxytubercidin, by mouse kidney. Using
[14C]tetraethylammonium
bromide ([14C]TEA) as
a substrate, we tested several renal cell lines for a
nucleoside-sensitive OCT. American opossum kidney proximal tubule cells
(OK) express a cimetidine-sensitive and metabolic-dependent ability to
efflux TEA. Other classic OCT inhibitors and several nucleosides also
inhibit TEA efflux by these cells in a manner reflecting structural
specificity for the carrier. Inhibition of OCT by nucleosides is not a
universal feature of OCTs, since TEA transport mediated by cloned rat
kidney OCT2 in the Xenopus laevis
oocyte system was not inhibited by the same nucleosides. In conclusion,
OK cells appear to possess an OCT that may also transport some
nucleosides by a novel carrier.
OK cells; tetraethylammonium; 2'-deoxytubercidin; cimetidine; transport
 |
INTRODUCTION |
MAMMALIAN KIDNEYS have the ability to actively remove
substances from circulating renal blood and transport them into the lumen of proximal tubules by a process called "renal secretion." In some cases the process is efficient such that 100% of the substance presented to the kidney is cleared from blood in a single passage, i.e., the renal plasma clearance equals the renal plasma flow. The
major, known renal secretory systems are pleiotropic and have been
placed into two distinct categories due to the nature of their
substrates and inhibitors: the organic cation transporter (OCT) and the
organic anion transporter (OAT) systems (18, 19). Classic substrates
for the OCT are tetraethylammonium (TEA) and N1-methylnicotinamide
(NMN), whereas p-aminohippurate (PAH)
and methotrexate are examples of substrates for the OAT. Classic
inhibitors are cimetidine (CIM), cyanine 863, and quinidine for the OCT
and probenecid for the OAT. Recently, three OCTs and two OATs have been
cloned from rat kidney, proving the existence of two classes of
carriers long ago predicted using pharmacological approaches. The OCTs
have considerable amino acid sequence homology as well as some homology
with the OATs (7, 16, 21, 23, 25, 30).
Some nucleosides are also secreted by human [deoxyadenosine
(dAdo), 2-chlorodeoxyadenosine (CldAdo), azidothymidine (AZT)] (9, 10, 17) and mouse [dAdo, 2'-deoxytubercidin
(dTub)] kidneys (10, 11). The secretion of dTub by mouse kidney
was shown to involve an OCT because:
1) secretion of dTub and TEA, but
not PAH, was inhibited in vivo by CIM (15);
2) dTub inhibited the active uptake
of TEA, but not PAH, by mouse kidney slices (11); and
3) organic cations, but not organic
anions, inhibited the active uptake of dTub by mouse kidney slices
(11). Thus we hypothesize that an OCT exists that also recognizes some
nucleosides, in particular dTub. To test this hypothesis and to obtain
a pure source for the putative carrier, we tested several renal cell lines for their ability to transport TEA by a mechanism inhibitable by
dTub or CIM. Among the cell lines examined, OK cells clearly and
reproducibly demonstrated an efflux mechanism consistent with active
secretion at the apical membrane. This report summarizes our findings
regarding this transporter to date. Some of these data have been
presented previously in abstract form (2, 3, 14).
 |
MATERIALS AND METHODS |
Cell lines. OK (opossum kidney
proximal tubule), Caki-1 (human clear cell carcinoma, renal primary),
LLC-PK1 (pig kidney proximal tubule), and MDCK (dog kidney distal tubule) cells were purchased from
the American Type Culture Collection (Rockville, MD). Renca (mouse
renal carcinoma) cells were obtained from Dr. Isaiah J. Fidler of the
Dept. of Cancer Biology, University of Texas M. D. Anderson Cancer
Center (28). OK,
LLC-PK1, and MDCK cells were
cultured at 37°C in DMEM with 10% (vol/vol) fetal bovine serum.
Caki-1 and Renca cells were cultured in McCoy's 5a and Eagle's MEM, respectively.
Uptake and efflux measurement. The OK
cells were cultured in 12-well dishes for 10 days. For uptake
experiments, the monolayers were incubated in 600 µl of DMEM
containing [14C]TEA
(0.1 µCi/ml, 30 µM) at 37°C in the absence or presence of inhibitors (300 µM). After various incubation times, the media were
removed by aspiration, and the monolayers were washed twice with PBS
(pH 7.4) at 4°C. The cells were lysed with 500 µl of 1 N NaOH for
3 h. The lysates were removed, and the incubation wells were washed
twice with 250 µl of 1 N HCl. The lysates and wash solutions were
combined and transferred to 7-ml scintillation vials. After the
addition of 5 ml liquid scintillation fluid (Research Product
International, Mount Prospect, IL), radioactivity was determined in a
scintillation spectrometer (model LS 3801; Beckman Instruments,
Fullerton, CA).
For efflux experiments, the cell monolayers were preincubated in 600 µl DMEM containing
[14C]TEA (0.1 µCi/ml, 30 µM) at 37°C for 3 h or overnight. The incubation media were then removed, and the monolayers were washed twice with PBS
at 4°C. DMEM (500 µl) with or without inhibitors (300 µM) was
then added to each well. After various incubation times, 500 µl of
the media were transferred directly to scintillation vials for
determination of radioactivity as described above. For measurements of
the uptake and efflux of dTub, the cells were incubated in the same
manner using [3H]dTub
(0.1 µCi/ml, 1 µM). Uptake and efflux experiments using Caki-1,
LLC-PK1, Renca, and MDCK cells
were performed after 4-7 days of incubation.
Efflux inhibitors. After incubating OK
cells with [14C]TEA
(0.1 µCi/ml, 30 µM) for 3 h, efflux was measured after a 20-min
incubation in increasing concentrations of potential inhibitors with or
without a maximally effective concentration of CIM (1 mM). The
IC50 is the concentration that
inhibited the CIM-sensitive TEA efflux by 50% as determined by
graphical analysis.
Metabolic and other dependence of TEA
efflux. After loading OK cells with
[14C]TEA (0.1 µCi/ml, 30 µM) by overnight incubation, the efflux was measured as
described above under different conditions as follow. For effects of
temperature, the efflux was measured at 25°C and 4°C over a
60-min interval. The effects of pH were determined by incubating the
cells at 25°C for 10 min in DMEM at pH 6.5 (buffer by MES) and 7.4 and 8.1 (buffered by HEPES). The effects of metabolic inhibitors were
determined by incubating the cells for 60 min at 25°C in the
absence of glucose and in the presence of 10 mM deoxyglucose and 10 µM rotenone. ATP levels in the cells following 60-min incubation was
determined by high-pressure liquid chromatographic analysis of
neutralized, acid-soluble extracts (11).
Xenopus laevis oocytes and
[14C]TEA transport
measurements.
The plasmid containing the rOCT2 cDNA (from rat kidney cortex) was a
gift from Dr. John B. Pritchard (Laboratory of Pharmacology and
Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, NC). The coding sequence for rOCT2 was located
between the Not I and
Sal I sites of a pSPORT1 vector (Life
Technologies, Gaithersburg, MD) with the start site of RNA transcription positioned downstream from a T7 RNA polymerase promoter. To prepare a rOCT2 cRNA, the vector was made linear by digestion with
Hind III, after which an in vitro
transcription kit (T7 mMessage mMachine; Ambion, Austin, TX) was used
to synthesize the 5'-capped cRNA. X. laevis oocytes were collected, defolliculated manually, and injected with 50 nl of water per oocyte with or without rOCT2 cRNA
(0.4 ng/nl). Three days after injection, the oocytes were incubated in
uptake solution [100 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 1 mM
CaCl2, 10 mM HEPES (pH 7.4)]
containing 180 µM
[14C]TEA and 1.8 mM of
various compounds (except for CldAdo, 1 mM) for 90 min at 25°C.
Where indicated, deoxycoformycin (DCF, 1 µM) was added to the uptake
solution to inhibit adenosine deaminase (ADA). At the end of the uptake
period, the oocytes were washed three times in ice-cold uptake
solution, then lysed in 0.2% SDS with 0.2 N NaOH, and the
radioactivity was determined by liquid scintillation counting as
described above.
Data analysis. Statistical analyses
were performed by the StatMost statistical analysis and graphics
program (Dataxiom Software, Los Angeles, CA). Data are means ± SE.
Significance for mean differences was determined by the unpaired
Student's t-test.
Materials.
[14C]TEA (3.36 mCi/mmol) was purchased from DuPont-NEN (Boston, MA);
[3H]dTub (9.3 Ci/mmol)
was from Moravek Biochemicals (Brea, CA); and
[14C]inulin (4.36 µCi/mg) was from ICN Pharmaceuticals (Irvine, CA). Nonradioactive
dTub was synthesized in our laboratory from tubercidin (Sigma Chemical,
St. Louis, MO) as previously described (20). Dideoxyribonucleosides
were purchased from ICN Biochemicals (Cleveland, OH). Other chemicals
were obtained from Sigma Chemical.
 |
RESULTS |
Uptake of TEA and dTub by renal cell lines in
culture. In seeking an efflux carrier that may be
expressed on the apical membrane of a renal tubular cell, we reasoned
that inhibitors might increase the "apparent uptake" at or near
steady state. Thus we measured the overnight uptake of a classic OCT
substrate, [14C]TEA,
in the presence and in the absence of CIM or dTub (Table 1). The uptake was enhanced by CIM in OK
and Caki-1 cells, whereas the uptake was inhibited by CIM in the other
cell lines. Enhanced uptake of TEA produced by dTub was significant in
OK cells, whereas the trend was toward inhibition of uptake in the
other cell lines examined. Thus OK cells represent a source of a
putative TEA, and perhaps dTub, carrier that is sensitive to
CIM, consistent with earlier data in mouse kidneys (11).
[3H]dTub uptake was
significantly enhanced by CIM, albeit biologically insignificant, in OK
and LLC-PK1 cells. The increased
uptake was most apparent in Caki-1 cells, for which the uptake after a
24-h incubation in the presence of CIM was increased to 212% of
control. This effect of CIM probably reflects inhibition of dTub efflux from the cells, since efflux of dTub was inhibited by CIM in this cell
line (data not shown).
To further investigate the nature of TEA uptake by OK cells, we
measured the uptake as a function of time in the presence and absence
of CIM or dTub (Fig. 1). The uptake of TEA
by OK cells is relatively rapid for this compound, which is generally
excluded from cells in the absence of a carrier, and steady state is
achieved after 30-min incubation. Although the uptake was inhibited by CIM at the earliest times examined (5 and 30 min), there was no inhibition by dTub at these times. Both compounds enhanced the uptake
after 3-h incubation. These results suggest that both uptake and efflux
are sensitive to CIM, whereas only the efflux is inhibited by dTub. To
determine possible influences of these compounds on efflux per se, the
following experiments were performed.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of cimetidine (CIM) and 2'-deoxytubercidin (dTub) on
[14C]tetraethylammonium
([14C]TEA) uptake by
OK cell monolayers. OK cells were incubated in
[14C]TEA (0.1 µCi/ml, 30 µM) in absence ( ) or presence of 300 µM CIM ( )
or dTub ( ) at 37°C. Data shown are means ± SE obtained from
2 experiments, each performed in triplicate.
[14C]inulin ( ) (0.1 µCi/ml, 30 µM) was used to indicate restriction of uptake to the
extracellular space.
|
|
Efflux of TEA by OK cells. Since the
apparent enhanced uptake of TEA seen after 24-h incubation in the
presence of CIM or dTub could reflect increased uptake, decreased
efflux, or both, we tested effects of these compounds on egress of this
substrate from the cells. The cells were loaded with
[14C]TEA by overnight
incubation with or without the presence of CIM or dTub. The efflux was
then measured in the presence or absence of CIM or dTub (Fig.
2). Efflux from the cells is gradual and almost complete in controls within a 1-h incubation period. During this
time, both CIM and dTub inhibit the efflux. These observations suggest
the presence of a TEA efflux mechanism in OK cells that is sensitive to
CIM and dTub. To determine whether dTub and CIM inhibit the same or
different carriers, dose-response effects of dTub were determined in
the absence or presence of a maximally effective concentration of CIM
(1 mM, Fig. 3). As shown in Fig. 3, dTub
did not add to the inhibition produced by CIM, consistent with
inhibition at a common site.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of CIM and dTub on efflux of
[14C]TEA. After
overnight incubation in
[14C]TEA (0.1 µCi/ml, 30 µM) with or without 300 µM CIM or dTub, efflux from OK
cells into media was measured in absence ( ) or presence of 300 µM
CIM ( ) or dTub ( ).
|
|

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 3.
Inhibition of CIM-sensitive TEA efflux by dTub. OK cells were incubated
in media containing
[14C]TEA (0.1 µCi/ml, 30 µM) for 3 h. Efflux of TEA into media was measured after
a 20-min incubation in increasing concentrations of dTub in presence or
absence of 1 mM CIM; , dTub; , dTub plus 1 mM CIM.
|
|
The IC50 values for inhibition of
the CIM-sensitive TEA efflux produced by compounds tested to date are
summarized in Table 2. None of these
inhibitors added to a maximally effective (1 mM) concentration of
CIM, as illustrated for dTub in Fig. 3. Classic OCT
inhibitors are the most potent, suggesting that this is an OCT that is
inhibited by some nucleosides such as dideoxycytidine (ddC), dTub, AZT,
and others. Of particular interest are the affinities of decynium 22, CIM, and NMN for this carrier, i.e., higher than those reported for
OCT1 and OCT2 (7, 16). The
pKa values for
CIM and dTub are 6.8 and 5.5, respectively (11, 13). According to
Henderson-Hasselbalch equation, the positive charged moiety of CIM
should be ~17-fold that of dTub at pH 7.4. If indeed the positive-charged form of CIM and dTub are the species that inhibit the
TEA efflux, then they may have similar affinities to the transporter. Interestingly, TEA is an extremely poor inhibitor of its own efflux. Some structural specificity for inhibition among nucleosides is also
apparent in the data summarized in Table 2. Namely, base specificity is
indicated by the differing potencies among dideoxyribonucleosides (ranging from 38 µM to more than 3,000 µM for cytosine, guanine, thymine, adenine, and hypoxanthine); and among the arabinosyl derivatives of guanine, cytosine, and adenine. Some sugar specificity is also suggested by the differing effects of the dideoxyribosyl and
arabinosyl derivatives of cytosine. Surprisingly, the adenine derivatives known to be secreted by kidney (dAdo, CldAdo) are rather
poor inhibitors of the efflux, compared with dTub, AZT, and some of the
dideoxyribonucleosides. Nitrobenzylthioinosine, a potent inhibitor of
the equilibrative, facilitated diffusion nucleoside transporter, also
inhibited TEA efflux by the OK cells; however, the order of potency (25 µM) is suggestive of a carrier other than that known as the "es
nucleoside transporter" that is ~1,000-fold more sensitive (1). It
should be mentioned that the intracellular concentrations of these
inhibitors may differ from that in the medium such that the actual
affinities at the site of action may differ from the results shown in
Table 2.
Further characterization of the TEA efflux by OK
cells. The efflux is profoundly temperature-dependent
as illustrated by the reduced rate of efflux at 4°C vs. 25°C
(Table 3). Metabolic dependence of the
efflux is also shown with ATP depletion by rotenone and deoxyglucose.
This treatment reduced ATP levels to 5.1 ± 2.6% of the control
value that averaged 1.4 µmol/109
cells (data not shown). Qualitatively similar effects on ATP levels and
TEA efflux were observed using sodium cyanide or iodoacetamide and
deoxyglucose (data not shown). With the exception of iodoacetamide, these treatments did not reduce cell viability as assessed by trypan
blue dye exclusion, i.e., the reduced TEA efflux produced by rotenone
and deoxyglucose is not due to general cytotoxicity. There is only a
minor influence of omission of sodium on the efflux, suggesting that
the flux of TEA from inside to outside is not dependent
upon sodium or that the substitution of choline for sodium produces a
slight inhibition of efflux under these conditions. Finally, the efflux
of TEA is clearly not an organic cation/proton exchange mechanism,
since reduced pH failed to enhance the rate of efflux. In summary, the
CIM-sensitive efflux of TEA by OK cells under the conditions studied is
not coupled to proton exchange and is metabolically dependent and
largely independent of extracellular sodium.
View this table:
[in this window]
[in a new window]
|
Table 3.
Effect of temperature, metabolic inhibitors, absence of sodium, and pH
on efflux of [14C]TEA by OK cells
|
|
Nucleosides fail to inhibit the transport of TEA by a
cloned OCT. Recently, two OCTs have been cloned from
rat kidney: OCT1 (7) and OCT2 (16). These proteins have ~70%
homology, and, although subject to validation, they probably function
as basolateral carriers enhancing the uptake of TEA into the proximal
tubule cells. As with the TEA efflux shown herein, classic OCT
inhibitors inhibit the uptake induced by cRNAs from these cloned cDNAs
in the X. laevis oocyte translation
system (7, 16). The cRNA for the OCT2 enhances TEA uptake by frog
oocytes markedly, and the enhanced uptake is inhibited by quinidine and
verapamil (Fig. 4). However, none of the
nucleosides or nucleoside analogs tested showed significant effect on
the OCT2 cRNA-dependent uptake, including dTub, AZT, and ddC, which
were all active toward TEA efflux by OK cells (Table 2).
In short, this finding suggests that inhibition of TEA transport by
nucleosides is not a phenomenon common to all OCTs. Additionally, in
similar experiments, the cRNA for OCT2 had no effect on the uptake or
efflux of [3H]dTub by
frog oocytes (experiments not shown), indicating that it is not a
carrier for this nucleoside that is secreted by mouse kidney (10, 15).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4.
Organic cation transporter OCT2 cRNA enhanced uptake of TEA by
X. laevis oocytes. X. laevis oocytes (15-20 per group) were injected
with 50 nl (20 ng) rOCT2 cRNA per oocyte. Control group was injected
with 50 nl of water. Three days after injection, oocytes were incubated
in 180 µM [14C]TEA
with 1.8 mM various inhibitors [except for 2-chlorodeoxyadenosine
(CldAdo), which was present at 1 mM] for 90 min. Oocytes were
then washed and lysed as described in MATERIALS AND
METHODS. Uptake of
[14C]TEA was
determined by liquid scintillation spectrometry. DCF, deoxycoformycin;
AZT, azidothymidine; ddC, dideoxycytidine; AraC, adenine arabinoside.
* Significantly different from control,
P 0.05, as determined by unpaired
Student's t-test.
|
|
 |
DISCUSSION |
The mammalian kidney plays a major role in maintaining an extracellular
fluid compatible with life. One mechanism by which the kidney
accomplishes this task is via secretory systems that improve the
elimination of toxins and drugs, transporting solutes from the blood
and extracellular space across the tubular epithelia into the lumen of
the nephrons. Barriers exist, therefore, on either side of the tubular
epithelia such that solutes must enter the cell via the basolateral
membrane, traffic inside the cell, and be transported into the lumen
across the apical membrane. Characteristics of the putative carrier
proteins involved at the basolateral and apical membrane are summarized
in a recent classic textbook of pharmacology (8). With regard to
organic cations, the active transport is thought to occur at the apical
membrane; however, mediated movement of model compounds such as TEA
occurs in membrane vesicles prepared from either the basolateral or
apical membranes. Passage across apical membranes is primarily via a proton exchanger, being enhanced by increasing hydrogen ion
concentrations. The gradient for protons, from lumen to intracellular
fluid, is favorable in the forming urine to drive this
exchange mechanism. This proton/organic cation exchanger has been
demonstrated in experiments using various renal brush-border membranes,
rabbit proximal tubules, and cultured OK cells (4, 29). In the latter case, the rapid uptake (30 s) of TEA by OK cells was studied and shown
to be influenced by a proton gradient (29). The mediated movement of
organic cations by basolateral membranes is not coupled to proton
exchange. The cDNAs encoding some of these transporter proteins have
been recently cloned from kidneys of rat, human, mouse, and rabbit as
well as a pig kidney cell line (5, 6, 7, 12, 16, 22, 24, 27, 30). The
OCTs are ~70% homologous to one another and are ~30% homologous
to the rat OAT (21, 23, 25). Studies reported to date suggest that
these carrier proteins reside on the basolateral membrane, i.e., they
serve to facilitate the uptake of organic cations into the cell, with
one exception. That is, OCT2 is said to be localized to the apical
membranes of distal tubular cells (5, 6), an unexpected finding since OCT has traditionally been considered a proximal tubular function. However, as seen herein (Table 1), there is a CIM-sensitive carrier for
TEA in the dog kidney distal tubular cell line, MDCK. It has been
suggested that the distal tubular location may serve to reabsorb some
cations such as dopamine that is synthesized in the proximal tubules
(5). Recently, a human fetal liver proton/organic cation antiporter was
cloned (hOCTN1, Ref. 26) which is also expressed in kidney. Metabolic
dependence of TEA uptake was demonstrated in hOCTN1 transfected cells,
and the amino acid sequence has a sugar transporter protein signature
and a nucleotide binding site (26); however, the possible relevance of
this TEA carrier to that reported herein is unknown.
Our interest in OCT arose from measurements of Ado and dAdo in the body
fluids of a child with severe combined immunodeficiency disease (ADA
deficiency) and in cancer patients treated with the ADA inhibitor, DCF
(10). The renal handling of Ado and dAdo is paradoxically different,
i.e., Ado undergoes a net reabsorption, whereas dAdo is secreted. The
renal secretion of dAdo was confirmed to occur in mice (10), and to
study the possible mechanism, we used the nonmetabolized and nontoxic
dAdo analog, dTub (11). This compound was also secreted by mouse
kidney, and the secretion was inhibited by CIM (15). Uptake of dTub
into mouse kidney slices was energy dependent, and the uptake was
inhibited by organic cations to a greater extent than by organic
anions. Conversely, dTub inhibited the uptake of TEA but not PAH by the
mouse kidney slices. Taken together, these criteria satisfy the
requirements of a substrate for "classic" OCT. Measurement of
dTub transport per se in cultured cells has been problematic, i.e., the
compound has sufficient lipid solubility that passive diffusion
frequently overshadows facilitated processes (unpublished
observations). This problem should not be interpreted to indicate the
situation that exists in the intact organ, i.e., it is difficult if not impossible to duplicate the whole organ condition where secretion of
dTub definitely occurs. In an effort to identify a dTub transporter, therefore, we used the impermeable solute, TEA. As such, our goal was
to identify a transporter in renal cells that was sensitive to dTub and
CIM. We further assumed that the carrier most likely to be novel would
be an efflux mechanism located on the apical membrane for reasons
described above. OK cells appear to possess such a carrier based upon
the preliminary characterization of TEA efflux reported herein. Namely,
the efflux is sensitive to CIM and other OCT inhibitors, and it is also
inhibited by dTub and other nucleosides (Fig. 3; Table 2). The
observation that dAdo and CldAdo fail to inhibit the TEA efflux by OK
cells (Table 2) suggests that this may not be the mechanism by which
they are secreted. Renal secretion of CldAdo in pediatric cancer
patients equaled the renal plasma flow, indicative of very efficient,
active transport (9). The observation that CIM failed to inhibit the secretion of dAdo in mice under conditions in which it did inhibit that
of TEA and dTub (15) also suggests that there may be other carriers for
dAdo and CldAdo. However, failure of TEA to inhibit its own efflux by
OK cells (Table 2) suggests that one should not interpret failure of
putative substrates to inhibit too broadly. Clearly, much is yet to be
learned regarding the various steps and carriers in the process of
renal secretion, and proof of the mechanisms involved will come only
after cloning and evaluation of all relevant carriers. To that end,
this work identifies a TEA efflux transporter in OK cells that is
consistent with an energy-dependent, nucleoside-sensitive process. Work
in progress using OK cell membrane vesicles will define further the
nature of this putative, novel carrier, e.g., ATP dependence,
nucleoside sensitivity.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
DK-41606 and 2-P30-CA-16672.
 |
FOOTNOTES |
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.
Address for reprint requests: J. A. Nelson, Dept. of Experimental
Pediatrics, Univ. of Texas M. D. Anderson Cancer Center, 1515 Holcombe
Blvd., Houston, TX 77030 (E-mail:
anelson{at}mdacc.tmc.edu).
Received 12 May 1998; accepted in final form 17 November 1998.
 |
REFERENCES |
1.
Cass, C. E.
Nucleoside transport.
In: Drug Transport in Antimicrobial and Anticancer Chemotherapy. New York: Dekker, 1995, p. 403.
2.
Chen, R.,
B. F. Pan,
M. Sakurai,
and
J. A. Nelson.
Organic cation and nucleoside transport (Abstract).
FASEB J.
12:
425,
1998.
3.
Chen, R.,
M. Sakurai,
B. F. Pan,
and
J. A. Nelson.
Nucleoside-sensitive and energy-dependent organic cation transport (OCT) by opossum kidney (OK) proximal tubule cells.
Proc. Am. Assoc. Cancer Res.
39:
610,
1998.
4.
Dantzler, W. H.,
O. H. Brokl,
and
S. H. Wright.
Brush-border TEA transport in intact proximal tubules and isolated membrane vesicles.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F290-F297,
1989[Abstract/Free Full Text].
5.
Gorboulev, V.,
J. C. Ulzheimer,
A. Akhoundova,
I. Ulzheimer-Teuber,
U. Karbach,
S. Quester,
C. Baumann,
F. Lang,
A. E. Busch,
and
H. Koepsell.
Cloning and characterization of two human polyspecific organic cation transporters.
DNA Cell Biol.
16:
871-881,
1997[Medline].
6.
Grundemann, D.,
J. Babin-Ebell,
F. Martel,
N. Ording,
A. Schmidt,
and
E. Schomig.
Primary structure and functional expression of the apical organic cation transporter from kidney epithelial LLC-PK1 cells.
J. Biol. Chem.
272:
10408-10413,
1997[Abstract/Free Full Text].
7.
Grundemann, D.,
V. Gorboulev,
S. Gambaryan,
M. Veyhl,
and
H. Koepsell.
Drug excretion mediated by a new prototype of polyspecific transporter.
Nature
372:
549-552,
1994[Medline].
8.
Jackson, E. K.
Diuretics.
In: Goodman and Gilman's The Pharmacological Basis of Therapeutics (9th ed.). New York: McGraw-Hill Health Professions Division, 1996, p. 685-713.
9.
Kearns, C. M.,
R. L. Blakley,
V. M. Santana,
and
W. R. Crom.
Pharmacokinetics of cladribine (2-chlorodeoxyadenosine) in children with acute leukemia.
Cancer Res.
54:
1235-1239,
1994[Abstract].
10.
Kuttesch, J. F., Jr.,
and
J. A. Nelson.
Renal handling of 2'-deoxyadenosine and adenosine in humans and mice.
Cancer Chemother. Pharmacol.
8:
221-229,
1982[Medline].
11.
Kuttesch, J. F., Jr.,
M. J. Robins,
and
J. A. Nelson.
Renal transport of 2'-deoxytubercidin in mice.
Biochem. Pharmacol.
31:
3387-3394,
1982[Medline].
12.
Lopez-Nieto, C.,
G. F. You,
K. T. Bush,
E. G. Barros,
D. R. Beier,
and
S. K. Nigam.
Molecular cloning and characterization of NKT, a gene product related to the organic cation transporter family that is almost exclusively expressed in the kidney.
J. Biol. Chem.
272:
6471-6478,
1997[Abstract/Free Full Text].
13.
McEvoy, G. K.
Miscellaneous GI drugs.
In: AHFS Drug Information 90. Bethesda, MD: American Society of Hospital Pharmacists, 1990, p. 1666.
14.
Nelson, J. A.,
R. Chen,
M. Sakurai,
and
B. F. Pan.
A nucleoside-sensitive, organic cation transporter (OCT) in opossum kidney (OK) cells (Abstract).
Toxicol. Sci.
42:
373,
1998.
15.
Nelson, J. A.,
J. F. Kuttesch, Jr.,
and
B. H. Herbert.
Renal secretion of purine nucleosides and their analogs in mice.
Biochem. Pharmacol.
32:
2323-2327,
1983[Medline].
16.
Okuda, M.,
H. Saito,
Y. Urakami,
M. Takano,
and
K. Inui.
cDNA cloning and functional expression of a novel rat kidney organic cation transporter, OCT2.
Biochem. Biophys. Res. Commun.
224:
500-507,
1996[Medline].
17.
Pioger, J. C.,
A. M. Taburet,
J. N. Colin,
S. Colaneri,
J. P. Fillastre,
and
E. Singlas.
Pharmacokinetics of zidovudine (AZT) and its metabolite (G-AZT) in healthy subjects and in patients with kidney failure.
Therapie
44:
401-404,
1989[Medline].
18.
Pritchard, J. B.,
and
D. S. Miller.
Renal secretion of organic anions and cations.
Kidney Int.
49:
1649-1654,
1996[Medline].
19.
Pritchard, J. B.,
and
D. S. Miller.
Mechanisms mediating renal secretion of organic anions and cations.
Physiol. Rev.
73:
765-796,
1993[Free Full Text].
20.
Robins, M. J.,
J. S. Wison,
and
F. Hansske.
Nucleic acid related compound. 42. A general procedure for the efficient deoxygeneration of secondary alcohols. Regiospecific and stereoselective conversion of ribonucleosides to 2'-deoxynucleosides.
J. Am. Chem. Soc.
105:
4065-4095,
1983.
21.
Saito, H.,
S. Masuda,
and
K. Inui.
Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney.
J. Biol. Chem.
271:
20719-20725,
1996[Abstract/Free Full Text].
22.
Schweife, N.,
and
D. P. Barlow.
The LX1 gene maps to mouse chromosome 17 and codes for a protein that is homologous to glucose and polyspecific transmembrane transporters.
Mamm. Genome
7:
735-740,
1996[Medline].
23.
Sekine, T.,
N. Watanabe,
M. Hosoyamada,
Y. Kanai,
and
H. Endou.
Expression cloning and characterization of a novel multispecific organic anion transporter.
J. Biol. Chem.
272:
18526-18529,
1997[Abstract/Free Full Text].
24.
Simonson, G. D.,
A. C. Vincent,
K. J. Roberg,
Y. Huang,
and
V. Iwanil.
Molecular cloning and characterization of a novel liver-specific transport protein.
J. Cell Sci.
107:
1065-1072,
1994[Abstract/Free Full Text].
25.
Sweet, D. H.,
N. A. Wolf,
and
J. B. Pritchard.
Expression cloning and characterization of ROAT1.
J. Biol. Chem.
272:
30088-30095,
1997[Abstract/Free Full Text].
26.
Tamai, I.,
H. Yabuuchi,
J. Nezu,
Y. Sai,
A. Oku,
M. Shimane,
and
A. Tsuji.
Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1.
FEBS Lett.
419:
107-111,
1997[Medline].
27.
Terashita, S.,
M. J. Dresser,
L. Zhang,
A. T. Gray,
S. C. Yost,
and
K. M. Giacomini.
Molecular cloning and functional expression of a rabbit renal organic cation transporter.
Biochim. Biophys. Acta
1369:
1-6,
1998[Medline].
28.
Utsugi, T.,
C. P. Dinney,
J. J. Killion,
and
I. J. Fidler.
In situ activation of mouse macrophages and therapy of spontaneous renal cell cancer metastasis by liposomes containing the lipopeptide CGP 31362.
Cancer Immunol. Immunother.
33:
375-381,
1991[Medline].
29.
Yuan, G.,
R. J. Ott,
C. Salgado,
and
K. M. Giacomini.
Transport of organic cations by a renal epithelial cell line (OK).
J. Biol. Chem.
266:
8978-8986,
1991[Abstract/Free Full Text].
30.
Zhang, L.,
M. J. Dresser,
J. K. Chun,
P. C. Babbitt,
and
K. M. Giacomini.
Cloning and functional characterization of a renal organic cation transporter isoform (rOCT1A).
J. Biol. Chem.
272:
16548-16554,
1997[Abstract/Free Full Text].
Am J Physiol Renal Physiol 276(2):F323-F328
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society