Fine tuning of rabbit equilibrative nucleoside transporter activityby an alternatively spliced variant*

SHARON K. WU1,†, DAVID K. ANN2, KWANG-JIN KIM3, & VINCENT H. L. LEE1,4

1Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089-9121, USA,2Departments of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA 90089-9121,

USA, 3Departments of Medicine, Physiology and Biophysics, Molecular Pharmacology and Toxicology, Biomedical Engineering,

Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, CA 90089-9121, USA, and4Departments of Pharmaceutical Sciences and Ophthalmology, University of Southern California, Los Angeles, CA 90089-

9121, USA

AbstractThe full-length cDNA encoding an equilibrative nucleoside transporter (rbENT2) and its novel C-terminal variant, rbENT2A,were isolated from rabbit trachea. Rabbit ENT2 protein consists of 456 amino acid residues; rbENT2A is shorter by 41 residues.Both rbENT2 and rbENT2A transcripts are found in rabbit tissues including intestine, kidney cortex, kidney, and trachea, atvarying levels of expression. When transfected in a heterologous expression system—Madin Darby canine kidney (MDCK)epithelial cell line—both rbENT2 and rbENT2A were expressed. rbENT2 had a molecular mass of 49 kDa; rbENT2A had amolecular mass of 44 kDa. Clones of both transporters yielded functional proteins that were capable of mediating uridine uptakeand efflux without the needing to be coupled to a secondary ion (e.g. Naþ). Remarkably, rbENT2A displayed a higher affinity(Km ¼ 41mM) and a lower capacity (Vmax ¼ 0.6 nmol/mg protein/5 min) towards substrates than rbENT2 (Km ¼ 272.8mM,Vmax ¼ 1.26 nmol/mg protein/5 min). Pharmacological profiles showed that nitro-benzyl-mercapto-purine-ribose (NBMPR)potently inhibited 3H-uridine uptake mediated by rbENT2A, but not uptake mediated by rbENT2. The constitutive splicing,broad expression, markedly different kinetics, and distinct pharmacological characteristics of rbENT2A appear to act inconjunction with the wild type, rbENT2, to fine-tune basolateral nucleoside transport function in rabbit trachea.

Keywords: C-terminal variant, rbENT2, MDCK, basolateral nucleoside transport, alternatively spliced variant

Abbreviations: ENT, equilibrative nucleoside transporters; CNT, concentrative nucleoside transporters; NBMPR, nitro-benzyl-mercapto-purine-ribose; es, equilibrative sensitive; ei, equilibrative insensitive; RTEC, rabbit tracheal epithelialcells; RACE, rapid amplification cDNA ends; MDCK, Madin Darby Canine Kidney; BSA, bovine serum albumin; S-MEM,Ca2þ-free minimum essential medium; DNase I, Deoxyribonuclease I; DEPC, diethylpyrocarbonate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RPA, ribonuclease protection assay; RT-PCR, reverse transcription-polymerase chain reaction;DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PAGE, polyacrylamidegel electrophoresis; TMD, transmembrane domain; PKA, protein kinase A; PKC, potein kinase C

Introduction

Nucleosides (and nucleotides) play a crucial role as

the activated precursors in DNA and RNA synthesis

and participate in physiological regulation of various

biological processes (e.g. cardiac function and the

regulation of glycolysis). Although most cells are

equipped with the biosynthetic machinery necessary

ISSN 1061-186X print/ISSN 1029-2330 online q 2005 Taylor & Francis

DOI: 10.1080/10611860500403099

*The nucleotide sequences reported in this paper were submitted on 24 November, 2000 to the GenBanke/EMBL Data Bank withaccession numbers AF323951 (for rbENT2) and AF323952 (for rbENT2A).

Correspondence: V. H. L. Lee, Food and Drug Administration, 5515 Security Lane, Room 1023, Rockville, MD 20852, USA.Tel: 1 301 443 5149. Fax: 1 301 443 5245. E-mail: leev@cder.fda.gov

†Present address: Cardinal Health Inc., Biotechnology and Sterile Life Sciences, 9250 Trade Place, San Diego, CA 92126, USA.

Journal of Drug Targeting, September–November 2005; 13(8–9): 521–533

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to synthesize nucleosides via de novo pathways, the re-

uptake of nucleosides from and their efflux into

extracellular space are thought to be important for

homeostasis of nucleosides (Wang et al. 1997, Gati

et al. 1998, Cass et al. 1999). In addition, these

molecules can act as ligands in cell signaling via

diverse purinergic receptors (Koshiba et al. 1997,

Apasov et al. 1997). Hence, cytoplasmic and

extracellular pharmacological manipulation of nucleo-

sides has wide therapeutic implications.

At least two very different classes of transporters

mediate the shuttling of these hydrophilic nucleoside

molecules across cell membranes. The first class, the

Naþ-independent, equilibrative nucleoside transpor-

ters (ENT), are more ubiquitously distributed among

cell types than the second, the Naþ-dependent,

concentrative nucleoside transporters (CNT). In

equilibrative transport, nucleoside flux across a given

membrane is bi-directional, driven by concentration

gradients. In contrast, concentrative transporters,

which are coupled to an electrochemical gradient of

an ion (e.g. Naþ or Hþ), transport nucleosides against

the concentration gradient. Naþ-independent, ENT

are subdivided into three classes based on their

sensitivity to a specific inhibitor, nitro-benzyl-mer-

capto-purine-ribose (NBMPR) (Griffith and Jarvis

1996). Transporters of the equilibrative sensitive (es or

ENT1) type are inhibited by NBMPR at nanomolar

ranges, whereas transporters of the equilibrative

insensitive (ei or ENT2) type are affected at or above

micromolar concentrations of NBMPR (Griffith and

Jarvis 1996). Additionally, the ei-type transporters

display affinity for purine nucleobases like hypo-

xanthine, as well as both purine and pyrimidine

nucleosides, giving them highly desirable character-

istics that might prove to be useful in various

therapeutic situations (Osses et al. 1996, Crawford

et al. 1998). The newest identified member of this

family, ENT3, found in acidic, intracellular compart-

ment, is a broad selectivity and low affinity nucleoside

transporter that can also transport adenine (Baldwin

et al. 2005). While ENT3 transport activity is relatively

insensitive to NBMPR, dipyridamole, or dilazep, it is

strongly dependent on pH (Baldwin et al. 2005).

To date, biophysical evidence describing the

kinetics of basolateral nucleoside transport in rabbit

airways has largely been lacking. We recently obtained

evidence from primary cultured rabbit tracheal

epithelial cells (RTEC) that basolateral es/ei type

transport exhibits a biphasic dose response to

NBMPR inhibition (Wu et al. 2005). In this study,

we isolated full-length cDNAs encoding a constitutive

equilibrative nucleoside transporter (rbENT2) and a

novel C-terminal variant rbENT2A from rabbit

trachea. We then transfected Madin Darby canine

kidney (MDCK) cells with rbENT2 and rbENT2A

cDNAs to determine their expression and function-

ality as ENT. Surprisingly, the full-length rbENT2

and its spliced variant, which have two different

putative C-termini, exhibited a wide difference in

RNA expression, kinetic properties, and pharmaco-

logical profiles.

Materials and methods

Methods

Male, Dutch-belted pigmented rabbits, weighing 2.5–

3.0 kg, were purchased from Irish Farms (Los

Angeles, CA). The investigations utilizing rabbits

described in this report conform to the Guiding

Principles in the Care and Use of Animals (DHEW

Publication, NIH 80–23). Protease XIV, DNase I,

DEPC, protease inhibitor cocktail, Triton X-100, and

BSA were purchased from Sigma Chemical Co. (St

Louis, MO). Marathone cDNA Amplification kit was

purchased from Clontech (Palo, Alto, CA). QIAquick

Gel Extraction kit was purchased from QIAGEN Inc.

(Valencia, CA). Biotrans nylon membranes were

purchased from ICN (Irvine, CA). RNADectectore

Northern Blotting Kit and PCR DNA Biotinylation

Kit were purchased from KPL Inc. (Gaithersburg,

MD). BioMaxe MS X-ray films were purchased from

Kodak (Rochester, NY). pGEMw-T Easy Vector

System was purchased from Promega (Madison, WI).

MAXIscripte T7/SP6 kit, RPA IIIe kit, BrightStare

Psoralen-Biotin nonisotopic labeling kit, BrightStarw-

Pluse positively charged nylon membrane, and

BrightStare nonisotopic RNA detection system were

purchased from Ambion Inc. (Austin, TX). Multi-

welle six and 12 well plates, and other cell culture

supplies, were purchased from Becton Dickinson and

Company (Franklin Lakes, NJ). Trans-Blotw nitro-

cellulose membrane and DC protein assay were

purchased from Bio-Rad Laboratories (Hercules,

CA). Mouse monoclonal antibody HA.11 was

purchased from Covance (Princeton, NJ). Peroxi-

dase-conjugated AffiniPure donkey anti-mouse IgG

was purchased from Jackson ImmunoResearch Lab-

oratories, Inc. (West Grove, PA). Super Signalw West

Pico chemiluminescence substrate was purchased

from Pierce (Rockford, IL). [5,6-3H]-uridine

(45.2 Ci/mmole) was purchased from Moravek Bio-

chemicals (Brea, CA). Econosafew scintillation cock-

tail was purchased from Research Products

International (Mount Prospect, IL). Unless indicated

otherwise, all the reagents in this study were obtained

from Invitrogen Co. (Carlsbad, CA).

Isolation of rabbit tracheal epithelial cells

We have already reported detailed procedures for

growing RTEC monolayers at an air-interface on a

permeable support (Mathias et al. 1996). Briefly,

pigmented rabbits were euthanized with an overdose

of sodium pentobarbital solution (85 mg/kg) then

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the trachea was excised and incubated for 60 min in

a protease solution (0.2% bacterial protease type XIV

in S-MEM) at 378C in a humidified atmosphere of 5%

CO2 and 95% air. The trachea’s mucosal surface was

gently scraped with a sterile surgical scalpel blade. The

detached epithelial cells were mixed with a DNase I

solution (0.5 mg/ml DNase I and 10% FBS in S-

MEM) at 378C, and centrifuged at 210 £ g for 10 min

at room temperature (the same settings were used to

pellet cells from suspension in all steps below). Cells

were washed with S-MEM containing 10% FBS by

resuspending the pellet and subsequent filtration

through a 70mm cell strainer.

RNA isolation

Freshly isolated tracheal epithelial cells (5 £ 106

cells/200ml) were lysed with 1 ml of TRIzolw by

repetitive pipetting. Tracheal RNA was extracted per

manufacturer’s direction. The purity and integrity of

the isolated RNA were verified by the ratio of

absorbances observed at 260 and 280 nm and also

by 1.5% agraose gel electrophoresis (see below).

Reverse transcription (RT) of cDNA from rabbit

tracheal RNA

We annealed 1ml of oligo (dT)18 (500mg/ml) to

5mg/11ml of RNA in DEPC-treated water by

incubation for 50 min at 428C in 20ml total volume

comprised of 4ml of 5 £ first strand buffer, 10 mM

dithiothreitol, 0.5 mM dNTP mix, and 1ml (200

units) of SuperScripte II. To inactivate the synthesis

reaction, the solution was heated at 708C for 15 min.

Two units of RNase H were added and incubated at

378C for 20 min to remove RNA complementary to

cDNA.

Degenerate polymerase chain reaction (PCR)

We chemically synthesized a degenerate primer pair

corresponding to the highly conserved peptide

sequences SGQGLAG and DWLGRSLT, as revealed

by multiple sequence alignment in Expert Protein

Analysis System (ExPASy) proteomics server at the

Swiss Institute of Bioinformatics with several existing

ENT family members, including ENT1, ENT2 and

HNP36 from human and rat. The nucleotide

sequences of the degenerate primers were: primer 1,

50-AGY GGC CAG GGC CTR GCW GG-30 (sense

strand); and primer 2, 50-GTWAGG CTC CGK CCY

ARC CAR TC-30 (antisense strand), where K

represents T þ G; R, A þ G; W, A þ C þ G and Y,

C þ T. The PCR reaction mixture (50ml) contained

5% of the cDNA obtained from reverse transcription,

along with 20 mM Tris–HCl (pH 8.4), 50 mM KCl,

2.5 units of Taq polymerase, 0.5 mM mixed deoxy-

nucleotides (dNTPs), and 20mM degenerate primers.

The PCR reaction conditions were as follows: 948C

for 4 min, one cycle; denaturation at 948C for 1 min,

annealing at 658C for 1 min, and elongation at 658C

for 2 min, 30 cycles; and 728C for 7 min, one cycle.

Thirty microliters of the PCR reaction mixture were

electrophoresed in a 1% agarose gel containing

0.5mg/ml ethidium bromide, and visualized by UV

light. The DNA band corresponding to the predicted

size (,513 bp) was cut out and extracted using the

QIAquick Gel Extraction kit. The resultant DNA

fragment was ligated into a TOPOe TA cloningw

vector following the manufacturer’s directions. The

ligated cDNA was expanded by transformation of

E. coli DH5a competent cells. More than 50 clones

were analyzed by endonuclease (EcoR I) restriction to

select insert-containing colonies. The resultant plas-

mid was sequenced by infrared fluorescent dye-

labeled M13 primers (GeneMed Synthesis Inc.,

South San Francisco, CA).

Molecular cloning of rbETN2 and its splice variant

Double-stranded cDNAs were reverse transcribed

from rabbit tracheal RNA and then ligated to the

adaptoradapter provided with the Marathone cDNA

Amplification kit. The antisense primer (50-GCAGC-

AGATGGGGTTGAAG. AACTC-30) or the sense

primer (50-GGCAGCCTGTTTGGGCAGCTG

GG-30) with the adaptor primer AP1 (50-CCATCC-

TAATACGACTCACTATAGGGC-30), respectively,

were used to obtain the remaining 50- or 30-ends of

cDNAs. Nested 50- or 30-RACE PCR was performed

using the antisense primer (50-GGCGGGGAAGAC-

CGACAGGGTGA-30) or the sense primer (50- CAC-

CCTCTTCCTCAGCGGCCAGG-30), respectively,

with the adaptor primer AP2 (50- ACTCACTATAG-

GGCTCGAGCGGC-30). The PCR reaction con-

ditions were as follows: denaturation at 948C for

4 min, one cycle; 948C for 1 min, and 3 min elongation

at 728C, five cycles; 948C for 1 min, and 3 min

elongation at 708C, five cycles; 948C for 1 min, and

3 min elongation at 688C, 30 cycles; and 728C for

7 min, one cycle. The PCR product was separated by

1% agarose gel electrophoresis, followed by elution

from the gel and then ligation into the TOPOe TA

cloningw vector that was used to transform E. coli

DH5a. Multiple clones were selected and analyzed for

the sequence of each RACE product. The full-length

cDNAs for rbENT2 and rbENT2A were generated by

primers complementary to the 50 and 30-end

sequences of the cDNA obtained from 50- and 30-

RACE under the PCR condition of 948C for 4 min,

one cycle; denaturation at 948C for 1 min, annealing at

608C for 1 min, and elongation at 728C for 1 min, 30

cycles; and 728C for 7 min, one cycle. The final

amplicon was subcloned into the TOPOe TA

cloningw vector and sequenced by infrared fluorescent

dye-labeled M13 primers.

Rabbit tracheal equilibrative nucleoside transporter rbENT2A 523

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Northern blot analysis

Up to 30mg of total RNA isolated from tracheal cells

was fractionated by 1.5% agarose gel electrophoresis

and transferred to Biotrans nylon membranes by

capillary action in 20 £ SSC. RNA was cross-linked

to the membrane by irradiation with UV light. Blots

were prehybridized in RNADectectore formamide

hybridization buffer in the presence of 100mg/ml

salmon sperm DNA for 1 h at 428C. Hybridization

was carried out overnight in the same buffer contain-

ing 50 ng/ml of a biotinylated rbENT2 gene-specific

probe made using PCR DNA Biotinylation Kit. The

blots were then washed twice for 15 min in

2 £ SSPE/0.5% SDS at room temperature, followed

by two washes for 30 min in 0.2 £ SSPE/0.5% SDS at

558C. Visualization of the signal was carried out on

BioMax RS X-ray films using the RNADetectore

Northern Blotting Kit. To determine the levels of

RNA loading, the biotinylated probe was stripped off

by washing in 0.5% SDS for 10 min at 958C, and then

re-probed with a biotinylated glyceraldehyde-3-phos-

phate dehydrogenase (GAPDH) probe as described

above. Normalization of the signals was performed

with respect to the GAPDH level.

Tissue distribution by RT-PCR

Oligonucleotides for the simultaneous detection of

rbENT2 and rbENT2A transcripts were designed

using the Mac Vectorw software (Oxford Molecular

group, Oxford, UK). The nucleotide sequences of the

primers were: primer 1, 50-CGT GGG CAT CGT

CCT GTC C-30 (sense strand); and primer 2, 50-GCA

GCA GAT GGG GTT GAA G-30 (antisense strand).

The PCR reaction mixture (50ml) contained 5% of

the cDNA reaction product from the reverse

transcription process, 20 mM Tris–HCl (pH 8.4),

50 mM KCl, 0.5 mM dNTP mixture, and 2.5 units of

Taq polymerase in the presence of 20mM primers.

The PCR reaction conditions were as follows: 948C

for 4 min, one cycle; denaturation at 948C for 1 min,

annealing at 558C for 1 min, and elongation at 728C

for 1 min, 27 cycles; and 728C for 7 min, one cycle.

PCR products were separated on a 2% agarose gel

containing 0.5mg/ml ethidium bromide, and visual-

ized by UV light.

Tissue distribution by ribonuclease protection assays

(RPA)

A PCR fragment of rbENT2 cDNA (nucleotide

positions 619–1016 in rbENT2 (GenBanke acces-

sion AF323951)) was amplified by RT-PCR with a

pair of primers designed according to the sequence

upstream and downstream of the alternative splicing

domain. The PCR product was ligated into the

pGEMw-T Easy Vector System. A cut by Sph I, at the

50 end of the insert, linearized the plasmid, then

an antisense RNA probe was synthesized by in vitro

transcription with SP6 RNA polymerase (MAXI-

scripte T7/SP6 kit). The RNA probe was biotin-

labeled using the BrightStare Psoralen-Biotin

nonisotopic labeling kit. RPA was performed using

a RPA IIIe kit according to the manufacturer’s

protocol. A rbENT2 cRNA (600 pg) was also

included in the RPA as a control and size indicator.

After RNase digestion, the protected RNA fragments

were precipitated, separated on a 5% polyacrylami-

de/8 M urea gel, blotted onto a BrightStarw-Pluse

positively-charged nylon membrane, and then

immobilized by UV cross-linking. The protected

mRNA bands corresponding to the rbENT2 or

rbENT2A were detected using the BrightStare

nonisotopic RNA detection system, then the signal

was visualized on BioMaxe RS X-ray films.

DNA constructs

Full-length cDNAs encoding rbENT2 (GenBanke

accession AF323951) or rbENT2A (GenBanke

accession AF323952) were subcloned in the sense

orientation into the mammalian expression vectors

pcDNA3 (Invitrogene) and pSG5-HA (pSG5 vector

(Stratagenee) tagged with N-terminal HA). The

pcDNA3 vector was used to investigate whether the

addition of HA tag to rbENT2 and rbENT2A

isoforms affects functional activity. A pair of primers,

50-GGGAATTCGCGCGAGGAGACGCCCCG-30

and 50-CCGCTCGAGTCAGAGCAGGGCCTTGA-

AGAG-30, were used to create two enzyme cutting

sites (EcoR I-Xho I, underlined) at the two ends of

rbENT2 or its splice variant. The EcoR I-Xho I

fragment covering the open reading frame (nucleotide

positions 18–1388 in rbEN2; nucleotide positions

18–1265 in rbENT2A) was amplified by PCR and

cloned between EcoR I and Xho I sites of pcDNA3 or

pSG5-HA vectors digested with the same enzymes.

Transient transfection by DNA constructs

For transfection studies, MDCK (strain I) epithelial

cells were seeded at a density of 4.0 £ 104 cells/cm2 in

Multiwelle six well plates and grown for 1 day to

,70% confluence. On day 2, 1mg DNA in 0.1 ml

Opti-MEM media was mixed with an equal amount of

Opti-MEM containing 6mg LipofectAMIMEe and

allowed to equilibrate for 15 min at room temperature.

Following equilibration, 0.8 ml Opti-MEM media was

used to further dilute the mixture, which was then

applied to MDCK cells for 24 h. To terminate

transfection, the bathing media were replaced with

Dulbecco’s modified Eagle’s medium (DMEM)

supplemented with 10% fetal bovine serum (FBS),

100 units/ml penicillin and 100mg/ml streptomycin

solution.

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Western blot analysis

Transfected MDCK cells were washed once with

phosphate-buffered saline (PBS), and then harvested

by gentle scraping and placed into 0.5 ml of PBS

containing 0.5% SDS and 1% protease inhibitor

cocktail. Cells were centrifuged at 15,000 £ g for

10 min at 48C to remove nuclei and unbroken cells.

Supernatants containing membrane proteins (30mg)

were mixed with an equal volume of 2 £ SDS-PAGE

sample buffer (20% (v/v) glycerol, 4% (w/v) SDS,

20 mM Tris–HCl, 0.01% (w/v) bromophenol blue,

2% mercaptoethanol) and electrophoresed with 10%

(w/v) SDS-PAGE, followed by electroblotting to a

Trans-Blotw nitrocellulose membrane. Membrane

blots were incubated with mouse anti-HA monoclonal

antibody diluted by 1:1000 (v/v). Peroxidase-

conjugated AffiniPure donkey anti-mouse IgG at a

dilution of 1:50,000 (v/v) was used to visualize the

HA-tagged proteins on a SuperSignalw enhanced

chemiluminescence (ECL) substrate.

Uptake studies in the transfected MDCK cells

Cells were transfected in Multiwelle six well plates,

then detached with 0.05% trypsin-EDTA at 24 h post-

transfection and replated onto Multiwelle 12 well

plates. Cells were cultured for another 24 h for uptake

studies. 3H-uridine was selected as a substrate for all

uptake studies. MDCK cells at 48 h post-transfection

were washed once with sodium-free Ringer’s solution

(SFR, containing 116.4 mM choline chloride, 5.4 mM

KCl, 5.6 mM glucose, 0.8 mM KH2PO4, 0.8 mM

MgSO4, 1.8 mM CaCl2·H2O, and 25 mM choline

bicarbonate) and allowed to equilibrate for 20 min. To

initiate the uptake of labeled uridine, a dosing solution

containing 5mM 3H-uridine (2mCi/ml) in SFR

replaced the SFR. Cells were washed in a cluster

plate three times with fresh ice-cold SFR to terminate

uptake. Washed cells were lysed using 0.5 ml of 0.5%

Triton X-100. Twenty microliters of cell lysates were

taken for protein assay (DC protein assay). Five

milliliters of Econosafew scintillation cocktail were

added to the rest of each cell lysate sample, which were

then assayed for radioactivity using LS 1801 System

(Beckman Instruments, Inc., Irvine, CA).

Concentration dependency

Transfected MDCK cells were spiked with 5mM3H-uridine (2mCi/ml) in the presence of 5, 10, 20, 50,

100, 200, 400, 600 and 800mM of unlabeled uridine

in SFR.

Effect of NBMPR

Transfected MDCK cells were exposed to different

concentrations of NBMPR, ranging from 1 nM to

100mM, which were premixed with the dosing

solution containing 5mM 3H-uridine (2mCi/ml) in

SFR. To determine the NBMPR sensitivity of uridine

uptake process, the dose-response curve, produced by

non-linear regression analysis using the GraphPad

Prismw version 3 for Windows (GraphPad Software,

San Diego, www.graphpad.com), was and used to

estimate the IC50 value for NBMPR.

Substrate selectivity

Unlabeled physiological purines (e.g. adenosine,

guanosine and inosine), pyrimidines (e.g. cytidine,

thymidine and uridine) and a nucleobase hypo-

xanthine (500mM each), were present in the dosing

solution of 5mM 3H-uridine (2mCi/ml) in SFR.

Results

Molecular characteristics of rabbit equilibrative-insensitive

nucleoside transporters

A full-length rabbit equilibrative-insensitive (ei)

nucleoside transporter (designated rbENT2) and its

splice variant rbENT2A, which differs in its C-

terminus, were identified by RT-PCR based RACE

cloning strategy. Sequence analysis revealed both a

38-bp deletion and a 9-bp insertion in the alterna-

tively-spliced region of genomic introns/exons in the

rbENT2 mRNA generation process. The combi-

nation of the two changes in the alternatively-spliced

region produced a total number of base pairs that was

not a multiple of three. This resulted in a frame shift

on the open reading frame leading to a premature stop

codon (Figure 1). The rbENT2 cDNA (GenBank

accession no. AF323951) is 2145 bp long with an

open reading frame of 1371 bp (including the stop

codon), encoding a 456 amino acid protein with a

predicted molecular mass of 50 kDa. This open

reading frame is flanked by a 17-bp 50-untranslated

region and a 757-bp 30-untranslated region. The

rbENT2A cDNA (GenBank accession no.

AF323952), on the other hand, has an open reading

frame encoding a protein of 415 amino acids, which is

41 residues shorter than rbENT2. The molecular

mass of rbENT2A protein was predicted to be 44 kDa.

The deduced amino acid sequence (Figure 2) of

rbENT2 exhibits significant similarity to several

sequences in GenBanke, including Naþ-independent

nucleoside transporters (Jarvis and Young 1986,

Boleti et al. 1997, Griffiths et al. 1997, Yao et al.

1997), adenosine-pyrimidine nucleoside transporter

(LdNT1) from the protozoan parasite Leishmania

donovani (Vasudevan et al. 1998), and adenosine

transporter from Toxoplasma gondii (Chiang et al.

1999). rbENT2 shows ,90% identity and 90%

similarity in primary amino acid sequence to its

human and rat homologs, hENT2 and rENT2,

Rabbit tracheal equilibrative nucleoside transporter rbENT2A 525

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respectively. Moreover, rbENT2 is 45% identical to

rENT1 and hENT1. This suggests that rbENT2,

rENT2, and hENT2 may share structural similarity,

whereas the rbENT2 and rbENT2A sequences are

only 69% identical and 76% similar at the amino acid

level. Membrane topology of rbENT2 and rbENT2A

based on Kyte-Doolittle approach was predicted using

TMPrede (available in The ExPASY Molecular

Biology Server, www.expasy.ch). The rbENT2 was

hypothesized to have 11 transmembrane domains

(TMDs). The TMDs are connected by short

hydrophilic regions, with the exception of putative

TMDs 1 and 2, which are connected by an

extracellular loop, and putative TMDs 6 and 7,

believed to be linked by a large cytoplasmic loop

(Figure 3A). By contrast, the rbENT2A protein was

predicted to have six or seven TMDs (Figure 3B and

C). The first six putative TMDs of rbENT2A,

comprising the amino acid segment spanning from

N-terminus to the point of alternative splicing

(located between TMD 6 and TMD 7), are predicted

to have a topology profile that is identical to that of the

full-length rbENT2 protein (Figure 3). Further

investigation is needed to determine whether the

amino acid sequence in the alternatively spliced region

in rbENT2A forms a long C-terminal tail or supports

a novel putative TMD7. Both proteins contain the

same potential N-linked glycosylation sites (Asp-47,

Asp-56) on an extracellular loop between putative

TMDs 1 and 2 based on PPsearche (available in The

ExPASY Molecular Biology Server, www.expasy.ch).

In multiple species (Griffiths et al. 1997a,b, Yao et al.

1997), Asp-47/48 is a conserved N-linked glycosyla-

tion site in both ENT1 and ENT2 isoforms. In both

rbENT2 and rbENT2A, PPsearche also highlights

Ser-227 as a possible protein kinase A (PKA)

phosphorylation site, located on an intracellular loop

between putative TMDs 6 and 7.

Tissue distribution of rbENT2/rbENT2A mRNA in the

rabbit

We next assessed the tissue distribution pattern of

these genes. This novel splicing event was not unique

to tracheal tissues. Using Northern blot analysis, we

observed a band of ,1.8–2.0 kb corresponding to

the rbENT2/rbENT2A trachea mRNA (Figure 4A).

The rbETN2A mRNA was not easily resolved from

that of rbETN2, since these two mRNAs only differ in

28 nucleotides. Therefore, we performed RT-PCR to

characterize the expression intensities of rbETN2A

with respect to rbENT2.RT-PCR analysis (Figure 4B)

shows that rbENT2 and rbENT2A transcripts coexist

in various tissues, including small intestine, kidney

cortex, and kidney medulla, as well as trachea. DNA

sequence analysis confirmed that nucleotide

sequences of these RT-PCR products are identical to

the corresponding segments of tracheal rbENT2 and

rbENT2A. To rule out the possibility of any RT-PCR

artifacts, we performed RPA using an anti-sense

cRNA that hybridizes to the message, thus circum-

venting both reverse transcription and polymerase

chain reaction steps. Biotin-labeled, 513-nucleotide

long, antisense RNA probes were used to selectively

Figure 1. Diagram of rbENT2 primary transcript and the gene products produced by alternative splicing. Splicing event #1 (alternative

splicing pathway) in which the hatched box (p) is deleted to yield a 2116-bp rbENT2A message. Splicing event #2 (default splicing pathway) in

which the black box (B) is deleted to result in a 2145-bp rbENT2 message. Translation of the messages produces the rbENT2 and rbENT2A

isoforms, which are identical through the amino acid residue 289, but then diverge (Figure 2).

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detect mRNA fragments, corresponding to rbETN2

and rbENT2A, differing by 28-nucleotides in length

(Figure 4C). Consistent with the RT-PCR results, two

ribonuclease-protected fragments with the expected

sizes (397 and 368 nucleotides, respectively, for

rbENT2 and rbETN2A) were detected in the same

tissue samples, trachea through kidney, used for RT-

PCR testing. The overall level of rbENT2 transcript

*

*

Figure 2. Deduced amino acid sequence of rbENT2 and rbENT2A compared with human equilibrative nucleoside transporters hENT1

(Griffiths et al. 1997a) and hENT2 (Griffiths et al. 1997b), and the rat equilibrative nucleoside transporter rENT2 (Yao et al. 1997).

Alignment was performed using ClustalW (Thompson et al. 1994) service at the European Bioinformatics Institute (EBI). Identical amino

acids among the five sequences are shown in white on a black background. Spaces introduced to optimize the alignment are indicated by dashed

lines. Labeled solid lines over hENT1 (Griffiths et al. 1997a) and under rbENT2 indicate the TMDs of these two transporters predicted by

TMPred Server (Hofmann and Stoffel 1993). The numbers at the right indicate the amino acid positions in each sequence. The putative N-

glycosylation sites and the potential PKA phosphorylation site are indicated by asterisks and arrow, respectively.

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expression was similar across the examined tissues

(Figure 4B and C). By comparison, rbENT2A

transcript, in contrast, was expressed at a lower

intensity than rbENT2, in all examined tissues. It is

currently unknown, however, whether the difference

in mRNA expression levels translates to functional

diversity.

Detection of recombinant, epitope-tagged

rbENT2/rbENT2A proteins expressed in MDCK cells

To examine whether the engineered recombinant

rbENT2 and rbENT2A constructs were expressed in

MDCK cells as proteins with the expected sizes,

cDNAs encoding rbENT2 and rbENT2A were

individually subcloned into HA-tagged pSG5 vectors

(antibodies against native rbENT2 were not avail-

able). The HA tag, representing the influenza

hemaglutinin epitope YPYDVPDYA, was fused to

the N-terminal ends of the rbENT2 and rbENT2A

recombinant transporters. The tag, composed of a

twelve amino acid peptide, CYPYDVPDYASL, can be

recognized by a monoclonal antibody (mouse mono-

clonal antibody HA.11 (Covance)). The constructs

(rbENT2/pSG5.HA and rbENT2A/pSG5.HA)

encoding rbENT2 and rbENT2A were transfected

into MDCK cells for in vitro heterologous protein

expression. The HA-tagged recombinant rbENT2

and rbENT2A proteins were detected in immunoblots

and had apparent molecular masses of 49 and 44 kDa,

respectively (Figure 5). As expected, no band was

detected in the cell lysate from cells transfected with

parent vector (pSG5.HA), indicating that the HA-

tagged rbENT2 and rbENT2A recombinant trans-

porters were indeed expressed in MDCK cells.

Characterization of endogenous uridine uptake in

MDCK cells

We next examined the background endogenous

uridine uptake in confluent MDCK cell monolayers

Figure 4. Detection of rbENT2 and rbENT2A transcripts by Northern blot analysis (Panel A), RT-PCR (Panel B) and RPA (Panel C). Panel

A: Detection of ENT2 by Northern blot analysis in rabbit trachea and rat jejunum (10). The RNA blot was hybridized with a biotinylated

rbENT2 gene-specific probe. The blot was then stripped and rehybridized with a GAPDH probe to ascertain uniform RNA loading. RNA size

markers are indicated on the left, and the approximate sizes of the hybridized signals are indicated on the right. Panel B: Total RNA isolated

from RTEC, kidney medulla, kidney cortex, and intestine were reverse transcribed using oligo (dT)18 primer. The resulting cDNAs were

amplified by using a set of rbENT2 gene-specific primers as described in the Experimental Procedures section. The RT-PCR products were

separated by electrophoresis through 2% agarose gel and visualized by UV with ethidium bromide staining. DNA size (in bp) markers are 506,

396, 344, and 298. Panel C: Total RNA isolated from RTEC (50mg), kidney medulla (30mg), kidney cortex (30mg), and intestine (30mg)

were protected with 100 pg of the antisense cRNA probe and digested with RNaseT1/A before precipitation, and then separated by

polyacrylamide gel as described in the Experimental Procedures section. The expected sizes of the protected fragments are indicated.

nt, nucleotides.

*

*

* *

*

*

Figure 3. Predicted membrane topological model of rbENT2 and

rbENT2A. Using TMPred Server (28), rbENT2 topology (Panel A)

was predicted to have 11 TMDs, with an intracellular N-terminus,

an extracellular C-terminus, and a large intracellular loop between

TMDs 6 and 7. rbENT2A was predicted to have either 6 (Panel B)

or 7 TMDs (Panel C) with an intracellular N-terminus. The putative

N-glycosylation sites and the potential PKA phosphorylation site are

indicated by asterisks and arrow, respectively.

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grown in 12-well cluster plates, using [3H]-uridine as a

substrate. To determine whether uridine uptake by

MDCK cells was mediated by saturable Naþ-

independent transport processes, the time course of

5mM [3H]uridine uptake under Naþ-free conditions

was measured in the presence and absence of 1 mM

unlabeled uridine (Figure 6A). Endogenous uridine

uptake in MDCK cells under Naþ-free conditions was

significantly abolished in the presence of 1 mM

unlabeled uridine. This indicates that MDCK cells

do exhibit Naþ-independent, saturable uridine uptake

process(es). To further characterize the specific type of

Naþ-independent nucleoside transport processes in

MDCK cells, we used NBMPR to differentiate

between functionally distinct es and ei subtypes of

ENT (Griffith and Jarvis 1996). The dose-response

curve of NBMPR shows the NBMPR sensitivity of

Naþ-independent, equilibrative nucleoside transport

processes in untransfected MDCK cells (Figure 6B).

The IC50 value for NBMPR was 24.3 nM, suggesting

the presence of an NBMPR-sensitive nucleoside

transport process in native MDCK cells. Thus, all

subsequent functional studies in transfected MDCK

cells were carried out in the presence of 100 nM

NBMPR in SFR. These conditions exclude Naþ-

dependent and Naþ-independent es types of nucleo-

side transport processes.

Functional characterization of rbENT2 and rbENT2A in

transiently-transfected MDCK cells

Constructs (rbENT2/pcDNA3 or rbETN2A/pcDNA3,

encoding rbENT2 or rbENT2A, respectively) were

transfected into MDCK cells, and the kinetic para-

meters and pharmacological profiles of uridine uptake

were examined. Time course of 5mM [3H]uridine

uptake by rbENT2 or rbENT2A-transfected MDCK

cells was linear for 15 min (data not shown). Hence,

5 min uptake studies were performed to determine the

functional and pharmacological classification of the

heterologously-expressed nucleoside transporters. The

Km for uridine of rbENT2 was 272.8 ^ 31.9mM and

the Vmax was 0.6 ^ 0.1 nmol/mg protein/5 min

(Figure 7A). In contrast, rbENT2A had a higher

affinity (Km ¼ 41.6 ^ 9.8mM) and lower capacity

(Vmax ¼ 1.3 ^ 0.1 nmol/mg protein/5 min) than

rbENT2 (Figure 7B). NBMPR inhibits 3H-uridine

uptake mediated by rbENT2A, with an IC50 of 0.1mM,

but not by rbENT2 (IC50 ¼ 200mM). At NBMPR

concentrations where rbENT2 still retained 100% of its

activity, rbENT2A activity was completely inhibited

(Figure 8). These data suggest that the recombinant

rbENT2 protein was NBMPR-insensitive. However,

the pharmacological profile of rbENT2A towards to

NBMPR changed to “es-type-like”. The rbENT2 data

herein are comparable to the pharmacological profile of

NBMPR, characterized in our recent studies of

basolateral uridine uptake in primary cultured RTEC

(IC25 ¼ 0.2mM, IC75 ¼ 270mM) (Wu et al. 2005).

Figure 5. Detection of recombinant, HA-tagged, rbENT2 and

rbENT2A proteins expressed in MDCK cells by Western blot

analysis. Cell membrane proteins were prepared from MDCK cells

transiently transfected with HA-tagged rbENT2 and rbENT2A

DNA constructs. Samples (20mg/lane) were subjected to 10% SDS-

polyacrylamide gel electrophoresis and transferred to a

nitrocellulose membrane. Membrane blot was incubated with

mouse anti-HA monoclonal antibody [1:1000 (v/v) dilution],

followed by wash and incubation with a peroxidase-conjugated

AffiniPure donkey anti-mouse IgG at a dilution of 1:50,000 (v/v).

The resulting signals were visualized on X-ray films by ECL. The

apparent molecular masses of the protein bands are indicated by

arrows on the right, and the positions of molecular-mass markers (in

kDa) are indicated on the left.

Figure 6. Characterization of endogenous equilibrative nucleoside

transport in untransfected MDCK cells. Panel A: Time course of

5mM [3H]-uridine uptake by MDCK cells under Naþ-free

condition. [3H]-uridine uptake was measured at 15-min intervals

in the absence (A) and presence (B) of 1 mM unlabeled uridine.

Points represent mean ^ sem, n ¼ 6. Panel B: Effect of NBMPR on

5mM [3H]-uridine uptake by MDCK cells under Naþ-free

condition. [3H]-uridine uptake was measured at 5 min as a

function of NBMPR concentrations. IC50 ¼ 24.3 ^ 1.1 nM was

estimated by non-linear curve fitting algorithms using GraphPad

Prism 3. Points represent mean ^ sem, n ¼ 6.

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We examined the effects of endogenous nucleosides

on [3H]uridine uptake by rbENT2- and rbENT2A-

transfected MDCK cells to determine their substrate

specificity. As shown in Figure 9, [3H]-uridine uptake

by these two variants was significantly inhibited by

40–90% in the presence of unlabeled nucleosides,

including guanosine, adenosine, inosine (purine

nucleosides); uridine, thymidine, cytidine (pyrimidine

nucleosides); and hypoxanthine (purine nucleobase, a

known ENT2 substrate). Thus, rbETN2 and

rbENT2A were broadly selective for physiological

purine and pyrimidine nucleosides.

Discussion

rbENT2A represents a novel variant of equilibrative-

insensitive (ei) nucleoside transporter

Alternative splicing of the primary transcript repre-

senting rbENT2 pre-mRNA produces two homo-

logous isoforms, rbENT2 and rbETN2A, differing in

their C-termini. Northern blot analysis with an

rbENT2-specifc cDNA probe demonstrated the

presence of a message approximately 2.0 kb in size,

consistent with the full-length rbENT2 cDNA. The

higher sensitivity of RPA and RT-PCR analyses were

required to confirm the presence of the alternatively

spliced variant, rbENT2A, whose expression was

Figure 8. Effect of NBMPR on 5mM [3H]-uridine uptake by

MDCK cells transiently transfected with pcDNA3/rbENT2 and

pcDNA3/rbENT2A constructs. [3H]-uridine uptake was measured

at 5 min as a function of NBMPR concentrations under Naþ-free

condition. NBMPR (0.1mM) was present in all assays to inhibit

endogenous es-type transport activity in these MDCK cells. IC50

values of NBMPR for rbENT2- (†) and rbENT2A-mediated

uridine uptake (O) were 200.3 ^ 7.8mM and 0.1 ^ 0.004mM,

respectively, estimated by non-linear curve fitting algorithms using

GraphPad Prism 3. Points represent mean ^ sem, n ¼ 6.

Figure 7. Concentration dependency of 5mM [3H]-uridine uptake

by MDCK cells transiently transfected with pcDNA3/rbENT2

(PanelA) and pcDNA3/rbENT2A (PanelB) constructs. [3H]-uridine

uptake was measured at 5 min as a function of unlabeled uridine

concentrations ranging from 0 to 800mM. NBMPR (0.1mM) was

present in all assays to inhibit endogenous es-type transport activity in

these MDCK cells. The rbENT2- or rbENT2A-mediated uridine

uptake (–) was calculated as the difference between the uptake data

observed in MDCK cells transfected with pcDNA3/rbENT2 (†) or

pcDNA3/rbENT2A (O) and those in MDCK cells transfected with

pcDNA3 alone (A), as shown in Panels A and B, respectively. Points

represent mean ^ sem, n ¼ 6.

Figure 9. Substrate selectivity of various nucleosides on 5mM

[3H]-uridine uptake by MDCK cells transiently transfected with

pcDNA3/rbENT2 and pcDNA3/rbENT2A constructs. [3H]-

uridine uptake was measured at 5 min in the absence (control)

and in the presence of 1 mM unlabeled nucleosides under Naþ-free

condition. B and A represent rbENT2- and rbENT2A-mediated

uridine uptake, respectively. NBMPR (0.1mM) was present in all

assays to inhibit endogenous es-type transport activity in these

MDCK cells. Asterisks(s) represent significant decrease in uridine

uptake compared to control (*p , 0.01) by one-way analyses of

variances, followed by Tukey’s procedure for contrasting multiple

group means. Each data point represents mean ^ sem, n ¼ 6.

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detected in a number of rabbit tissues. The variant

occurs in equal concentrations as the wild type in all

tissues studied, which suggests that the alternative

splicing event is constitutive in nature. This type of

expression is in contrast to other phenomena in which

the splicing event appears to be regulated by distinct

key physiological cues, in response to certain external

stimuli or developmental prompts in a growing

organism.

Epitope-tagged rbENT2 and rbENT2A transcripts

were efficiently translated into 49- and 44-kDa

proteins, respectively, in transfected MDCK cells.

This suggests that the endogenous rbENT2 and

rbENT2A messages can serve as a template for

protein synthesis. However, these proteins need to be

detected in native tissues/cells to confirm their

existence. To directly test the presence and spatio-

temporal distribution of nucleoside transporters by

Western blot analysis and immunohistochemistry

isoform-specific antibodies need to be developed.

Functional significance of C-terminus in rbENT2A

Alternative splicing of ENT1 variants has been

reported for the mouse homologues (Kiss et al.

2000, Handa et al. 2001). One of the variants encodes

for two additional amino acid residues in a large

intracellular loop, resulting in a lower functional

activity. The other has a distinct sequence in its 50-

untranslated region (Kiss et al. 2000). However, their

kinetic and pharmacological properties (as compared

to those of the wild-type, mENT1) were not

determined yet. Many reports of alternative splice

variants resulting in diverse consequences exist. These

include variants with similar activities e.g. 5-HT7

receptor (Heidmann et al. 1998), and glycine

transporter 1-c (Kim et al. 1994) and those that are

non-functional with respect to the wild type e.g.

CFTR lacking exon 9 (Pagani et al. 2000) and 5-HT6

receptor with a 289-bp deletion (Olsen et al. 1999).

On the other hand, some nonfunctional spliced

variants like the H1 receptor (GHR1-279) (Ross

et al. 1997) act as negative regulators of their wild

type. The hPepT1 regulating factor (Saito et al. 1997),

generated from the same pre-mRNA as hPepT1, acts

as a regulator of hPepT1 function as its name implies.

In this study, we report for the first time a novel

splice variant (rbENT2A) differing in its C-terminus

from the Naþ-independent, NBMPR-insensitive

nucleoside transporter (rbENT2). Alternative splicing

of rbENT2 led to lowered expression and es-like

functional properties, but has no effect on ion

independence. Our RT-PCR and RPA results reveal

that the rbENT2 transcript has a higher RNA

expression level than rbENT2A Expression of the

rbENT2A variant containing the novel C-terminus

produced nucleoside transporters with different

kinetic characteristics and divergent pharmacological

properties. The splice variant exhibited a higher

affinity (Km ¼ 0.04 mM) towards substrates than

rbENT2 (Km ¼ 0.27 mM) and other cloned ENT2s

(hENT2, 0.20 mM (Griffiths et al. 1997a, b); rENT2,

0.30 mM (Yao et al. 1997)). The splice variant

exhibited a lower capacity (Vmax ¼ 0.6 nmol/mg

protein/5 min) towards substrates than did rbENT2

(Vmax ¼ 1.3 nmol/mg protein/5 min). Interestingly,

rbENT2A behaves as an es type-like nucleoside

transporter, with an IC50 value of 0.1mM NBMPR.

The wild types rbENT2, hENT2 (Griffiths et al.

1997a,b) and rENT2 (Yao et al. 1997) were all

NBMPR-insensitive, and their IC50 values of

NBMPR were .1mM. Although rbENT2A possesses

higher sensitivity to NBMPR than other ENT2s,

rbENT2A is still less sensitive to NBMPR than

genuine es-type nucleoside transporters (hENT1,

IC50 ¼ 3.6 nM (Griffiths et al. 1997a, b); rENT1,

IC50 ¼ 4.6 nM (Yao et al. 1997)). Kinetic measure-

ments indicated that both rbENT2 and its splice

variant proteins were indeed capable of mediating

uridine uptake.

Using a chimera approach, Sundaram et al. (1998)

determined that TMDs 3–6 of hENT1/rENT1

transporters were responsible for the interactions of

the transporters with NBMPR and vasoactive drugs,

and were likely to form parts of the substrate

translocation channel. Additionally, domain swapping

methods narrowed down the major sites of NBMPR

interaction in chimeric nucleoside transporters to

TMDs 3–4 and TMDs 5–6 (Sundaram et al. 2001).

As a prelude to such investigation, the orientation and

membrane topology of rbENT2/2A transporter

proteins needs to be clarified. A C-terminal truncated

form of rbENT2, composed of the amino acid

sequence starting from N-terminus to the point of

alternative splicing (located after TMD6), will be

constructed to study its functionality and subcellular

distribution in truncated or chimeric recombination.

Conclusion

Molecular cloning of ENT2 isoforms was accom-

plished utilizing probes designed from conserved

regions of the ENT family. Functional characteriz-

ation of successful clones by a heterologous expression

system revealed evidence that a pair of proteins arises

from the same gene through alternative splicing. The

two transporters appeared to coexist in rabbit trachea

with distinct pharmacological profiles. This substan-

tiated the existence of a novel splice variant, rbENT2A

(es-like), which bears no identity to and little similarity

with any known Naþ-independent es nucleoside

transporter group. The rbENT2A arises by alternative

splicing of pre-mRNA encoding for rbENT2, an ei

type ENT.

Our functional studies suggested that rbENT2 and

rbENT2A may play a role in absorption, disposition,

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and elimination of nucleosides and nucleoside drugs

via ENT. With the molecular identification of these

nucleoside-transport proteins, an understanding of

the relationship between deduced primary amino acid

sequence and putative secondary topological infor-

mation provides a structural basis for the functional

differences among the two transporter isoforms.

Moreover, the development of a heterologous

expression system for production of recombinant

rbENT2/2A provides a better picture of the mechan-

isms of these two nucleoside transporters in rabbit

trachea. The information on functional kinetics and

substrate selectivity of rabbit tracheal ENTs obtained

in this study may be useful for the design of in vivo

targeting of nucleoside drugs and their delivery for the

treatment of pulmonary diseases.

Acknowledgements

We thank Dr Wei-Chiang Shen (University of

Southern California) for kindly providing MDCK

epithelial cell line (stain I) for heterologous expression

of the rbENT2 and rbETN2A genes. This work was

supported in part by the American Heart Association

Grant-in-Aid 9950442N (to K.-J.K.) and National

Institutes of Health Grants GM52812 (to V.H.L.L.),

HL38658 (to K.-J.K.), and HL64365 (to K.-J.K. and

V.H.L.L.).

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