HPLC reveals novel features of nucleoside and nucleobase homeostasis, nucleoside metabolism and nucleoside transport

Journal Pre-proof HPLC reveals novel features of nucleoside and nucleobase homeostasis, nucleoside metabolism and nucleoside transport

Reema A. Altaweraqi, Sylvia Y.M. Yao, Kyla M. Smith, Carol E. Cass, James D. Young PII:

S0005-2736(20)30072-9

DOI:

https://doi.org/10.1016/j.bbamem.2020.183247

Reference:

BBAMEM 183247

To appear in:

BBA - Biomembranes

Received date:

6 December 2019

Revised date:

16 February 2020

Accepted date:

18 February 2020

Please cite this article as: R.A. Altaweraqi, S.Y.M. Yao, K.M. Smith, et al., HPLC reveals novel features of nucleoside and nucleobase homeostasis, nucleoside metabolism and nucleoside transport, BBA - Biomembranes(2020), https://doi.org/10.1016/ j.bbamem.2020.183247

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© 2020 Published by Elsevier.

Journal Pre-proof HPLC reveals novel features of nucleoside and nucleobase homeostasis, nucleoside metabolism and nucleoside transport

Reema A. Altaweraqi1*, Sylvia Y.M. Yao1, Kyla M. Smith1, Carol E. Cass2, James D. Young1

From the Membrane Protein Disease Research Group, Departments of 1Physiology and 2

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Oncology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

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*Present address: Department of Biochemistry, King Saud University, Riyadh, Saudi Arabia

To whom correspondence should be addressed: Dr. James D. Young, Department of

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Physiology, 7-55 Medical Sciences Bldg., University of Alberta, Edmonton, Alberta, T6G

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2H7, Canada. Tel.: 780-492-5895; Fax: 780-248-1995; Email: [email protected]

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Journal Pre-proof Abstract Humans possess three members of the cation-coupled concentrative nucleoside transporter CNT (SLC 28) family, hCNT1-3: hCNT1 is selective for pyrimidine nucleosides but also transports adenosine, hCNT2 transports purine nucleosides and uridine, and hCNT3 transports both pyrimidine and purine nucleosides. hCNT1/2 transport nucleosides using the transmembrane Na+ electrochemical gradient, while hCNT3 is both Na+- and H+coupled.

By producing recombinant hCNT3 in Xenopus laevis oocytes, we have used

radiochemical high performance liquid chromatography (HPLC) analysis to investigate the

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metabolic fate of transported [3H] or [14C] pyrimidine and purine nucleosides once inside

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cells. With the exception of adenosine, transported nucleosides were generally subject to minimal intracellular metabolism. We also used radiochemical HPLC analysis to study the

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mechanism by which adenosine functions as a low Km, low Vmax permeant of hCNT1. hCNT1-producing oocytes were pre-loaded with [3H]uridine, after which efflux of

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accumulated radioactivity was measured in transport medium alone, or in the presence of hCNT1-mediated [3H]-efflux was

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extracellular non-radiolabelled adenosine or uridine.

stimulated by extracellular uridine, but inhibited by extracellular adenosine, with > 95% of the radioactivity exiting cells being unmetabolized uridine, consistent with a low

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transmembrane mobility of the hCNT1/adenosine complex. Humans also possess four members of the equilibrative nucleoside transporter ENT (SLC 29) family, hENT1-4. Of

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these, hENT1 and hENT2 transport both nucleosides and nucleobases into and out of cells,

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but their relative contributions to nucleoside and nucleobase homeostasis and, in particular, to adenosine signaling via purinoreceptors, are not known. We therefore used HPLC to determine plasma nucleoside and nucleobase concentrations in wild-type, mENT1-, mENT2- and mENT1/mENT2-knockout (KO) mice, and to compare the findings with knockout of mCNT3. Results demonstrated that ENT1 was more important than ENT2 or CNT3 in determining plasma adenosine concentrations, indicated modest roles of ENT1 in the homeostasis of other nucleosides, and suggested that none of the transporters is a major participant in handling of nucleobases.

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Journal Pre-proof Keywords: Nucleoside transporters, radiochemical high performance liquid chromatography,

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knockout mice, Xenopus laevis oocytes, nucleoside metabolism

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Journal Pre-proof 1. Introduction Nucleosides and nucleobases are important salvage metabolites, serving as precursors of DNA, RNA and high-energy compounds such as ATP [1]. The purine nucleoside adenosine is also a ubiquitous physiological signaling molecule [2]. Since most nucleosides are hydrophilic in nature, they require specialized nucleoside transporter proteins for passage across cell membranes. Cellular uptake of therapeutic anticancer and antiviral nucleoside analogs is mediated by the same nucleoside transport processes [1-3] Two structurally unrelated protein families are responsible for membrane transport of nucleosides

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in humans and other mammalian species: the Concentrative Nucleoside Transporter (CNT)

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family (SLC28 in humans) and the Equilibrative Nucleoside Transporter (ENT) family (SLC 29 in humans) [1]. CNTs mediate cation-coupled nucleoside import into cells, while ENTs

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mediate passive bi-directional transport of both nucleosides and nucleobases. There are

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three mammalian CNT isoforms (CNT1-3), and four ENT isoforms (ENT1-4). Present in epithelia and other specialized cells, and driven by Na+ (CNT1/2) and/or

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Na+/H+ electrochemical gradients (CNT3), CNT1 transports pyrimidine nucleosides and adenosine, CNT2 transports purine and uridine, and CNT3 transports both purine and

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pyrimidine nucleosides. Adenosine is an atypical CNT1 permeant, being transported with

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high apparent affinity, but low maximum velocity [4]. Broadly selective for both purine and pyrimidine nucleosides and nucleobases, and

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ubiquitously expressed across tissues, Na+-independent ENT1 and ENT2 transport nucleosides and nucleobases passively across plasma and intracellular membranes. With the exception of inosine, which is transported similarly by both transporters, ENT1 has higher apparent affinities for nucleosides than ENT2, while ENT2 has higher apparent affinities for nucleobases [5]. ENT3 also has broad permeant selectivity, and functions intracellularly in lysosomes [6].

ENT4 is found in plasma membranes and transports adenosine and

monoamines in the brain and heart [7-9]. With this complexity of distribution and function, it is unclear which of these transporters is/are the most important in nucleoside and nucleobase homeostasis.

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Journal Pre-proof The CNT and ENT protein families are structurally unrelated to each other, having very different membrane architectures and 3D-structures [10-12]. CNTs function by an elevator-type transport mechanism, whereas ENTs exhibit a different alternating access-type transport mechanism. In the former, the permeant binding site physically moves across the membrane [11]. In the latter, a centrally-located permeant binding site is alternately exposed to extracellular and intracellular membrane surfaces [12]. Over the past 25 years, there has accumulated a large body of work on the properties of recombinant nucleoside transporters produced in oocytes of Xenopus laevis. Oocytes lack

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endogenous nucleoside transport activity [13], but there is essentially no information on the

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metabolism of nucleosides in oocytes, and how such metabolism might impact experimentally measurements of influx or efflux of nucleosides. In the present study,

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radiochemical HPLC analysis was used to fill in this knowledge gap by determining the extent of intracellular nucleoside metabolism under conditions typical of nucleoside

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transport assays in Xenopus oocytes producing recombinant broad-specificity hCNT3. We

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also used radiochemical HPLC analysis and electrophysiology to confirm that adenosine functions as an inhibitor of the human CNT1 isoform (hCNT1), and to further investigate

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the mechanism of that inhibition.

In a final series of experiments to address the respective roles of ENTs and CNTs in

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nucleoside and nucleobase homeostasis, HPLC was used to investigate differences in plasma levels of nucleosides and nucleobases in wild-type (WT) mice, and in knockout (KO) mice

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deficient in mENT1, mENT2, mENT1/mENT2, or mCNT3.

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Journal Pre-proof 2. Materials and Methods 2.1. Heterologous expression of NTs in Xenopus oocytes Plasmid cDNAs of hCNT1 and hCNT3 (GenBank™ accession numbers U62968, AF305210, respectively) were linearized with Nhe1 and transcribed with T7 polymerase using the mMESSAGE mMACHINETM (Ambion, USA) in vitro transcription system. Template DNA was removed by digestion with RNase-free DNase1. Healthy stage V - VI oocytes were isolated by collagenase treatment (2 mg/ml for 2 hours) of ovarian lobes from female

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Xenopus laevis (Biological Sciences Vivarium, University of Alberta, Canada) that had been

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anaesthetized by immersion in 0.3% (w/v) tricaine methanesulfonate (pH 7.4). Frogs were humanely sacrificed following collection of oocytes in compliance with guidelines approved

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by the Canadian Council on Animal Care. The remaining follicular layers were removed by phosphate treatment (100 mM K2PO4) and manual defolliculation. Twenty-four hours after

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defolliculation, oocytes were injected with either 10 nl of water containing 1 ng/nl of capped

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RNA transcript, or the same volume of water alone. Injected oocytes were then incubated for 4 days at 18C in modified Barth’s solution (changed daily) (88 mM NaCl, 1 mM KCl,

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0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3, 10 mM HEPES, 2.5 mM sodium pyruvate, 0.1 mg/ml penicillin and 0.05 mg/ml gentamycin sulfate, pH 7.5)

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prior to the assay of nucleoside transport activity or oocyte extraction.

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2.2 Radioisotope Flux Studies

Radioisotope transport assays were performed in groups of 20 oocytes at 20°C using 3

H- or14C-labeled nucleosides (2.5 μCi/ml or 1 μCi/ml, respectively) in 200 μl transport

medium containing 100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES, pH 7.5.

Nucleoside uptake was determined at concentrations of 20 μM.

Radioactive nucleosides were obtained from Moravek Biochemicals (Brea, California). Following either 1-min or 30-min incubations, extracellular radioactivity was removed by six rapid washes in ice-cold Na+-free choline chloride (ChCl) transport medium (100 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES, pH 7.5). Individual oocytes were then dissolved in 1% (w/v) SDS solution for quantification of oocyte-associated radioactivity by liquid scintillation counting (LS 6000IC, Beckman Canada 6

Journal Pre-proof Inc., Canada). In adenosine uptake experiments, oocytes were pretreated (30 min at 20°C) with 1 μM 2’-deoxycoformycin to inhibit adenosine deaminase activity.

The 2’-

deoxycoformycin was a generous gift from Dr. W. Gati, Department of Pharmacology, University of Alberta. For efflux experiments, groups of 20 hCNT1 RNA-injected oocytes were preloaded with 10 μM [3H] uridine (2.5 μCi/ml) in 0.2 ml of NaCl transport medium at 20° for 30 min, followed by six rapid ice-cold washes in NaCl transport medium to remove extracellular 3H. One group of 20 oocytes was processed to determine the time-zero [3H] uridine content

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(typically 50-60 pmol/oocyte). Other groups of 20 oocytes were resuspended in 1 mM of

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NaCl transport medium (± 1 mM nonradioactive nucleoside) at 20° to initiate efflux. At predetermined time intervals, duplicate 10-μl samples of incubation medium were removed

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and counted for 3H.

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2.3 Electrophysiology

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For electrophysiological studies of recombinant hCNT1 produced in Xenopus oocytes, nucleoside-evoked membrane currents were measured at room temperature using a

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GeneClamp 500B oocyte clamp (Molecular Devices, Sunnyvale, CA) in the two-electrode, voltage-clamp mode [34]. The GeneClamp 500B was interfaced to an IBM-compatible PC

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via a Digidata 1322A A/D converter and controlled by pCLAMP software (version 9.0; Molecular Devices). The microelectrodes were filled with 3 M KCl and had resistances that

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ranged from 0.5 to 2.5 MΩ. Oocytes were penetrated with the microelectrodes and their membrane potentials were monitored for periods of 5–10 min. Oocytes were discarded when membrane potentials were unstable, or more positive than −30 mV. All steady-state current measurements were performed at a holding potential (Vh) of −50 mV. Current responses were generated by perfusing individual hCNT1-producing oocytes with 100 µM of uridine, 100 µM adenosine, or 100 µM each of uridine and adenosine in a sodium-containing transport medium containing 100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 8.5. Choline replaced Na+ in Na+-dependence experiments, and conditions involving adenosine included 2’-deoxycoformycin (500 nM) to inhibit adenosine deaminase activity. The same experiments were performed in control water-injected oocytes. Current signals were filtered at 20 Hz (four-pole Bessel filter) and sampled at 20-millisecond 7

Journal Pre-proof intervals. Results are given as means ± S.E.M. for 4–6 individual oocytes. The experiment was performed twice on oocytes from different frogs, yielding closely similar results. 2.4 NT KO mice Mice that were heterozygous for disruptions in either the mENT1, mENT2 or mCNT3 genes were purchased from Lexicon Pharmaceuticals, Inc. (The Woodlands, Texas). Schematic representations of the modified mENT1, mENT2 and mCNT3 genes are shown in Fig. 1. Those for mENT1 and mENT2 carried a retroviral insertion between exons 2 and

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3. That for mCNT3 carried a deletion of hCNT3 genomic sequence between and including exons 4 and 6. The heterozygotes were backcrossed for six generations into FVB/N mice

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(Taconic, Hudson, New York), and the resulting FVB/N mENT1, FVB/N mENT2 and

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FVB/N mCNT3 heterozygous mice were then crossed to derive the mENT1 homozygous KO strain, the mENT2 homozygous KO strain, and the mCNT3 homozygous KO strain.

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mENT1 homozygous KO mice were then crossed with mENT2 homozygous KO mice to generate a mENT1/mENT2 double homozygous KO strain.

These fully backcrossed

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mENT1 KO mice, mENT2 KO mice, mCNT3 KO mice, and mENT1/mENT2 double KO mice were backcrossed again into FVB/N every 30 generations to maintain genetic

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homogeneity [14]. FVB/N mice served as wild-type controls. Both female and male mice of 4–12 months in age were used in experiments, and were housed in ventilated cages in a

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barrier-maintained facility at the Cross Cancer Institute. Animal work reported here was approved by the Animal Care Committee of the Cross Cancer Institute, a facility of Alberta

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Health Services, Cancer Care, and all animals were handled in accordance with regulations of the Canadian Council on Animal Care. As illustrated in Fig. 2 for mCNT3, the genotypes of wild-type, mENT1 KO, mENT2, mENT1/2 double KO or mCNT3 KO mice were confirmed by PCR of genomic DNA using pairs of primers designed to identify the strains. Mice from each of the KO strains were viable, fertile and of normal physical appearance. 2.5 Preparation of Oocyte Extracts for HPLC Analysis Groups of twenty oocytes were collected after radioisotope flux studies and homogenized together in 500 μl of ice-cold hypotonic buffer (7.5 mM HPO4, 1 mM EDTA, pH 7.4), deproteinized by adding 30 μl ice-cold 70% w/w perchloric acid, and centrifuged

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Journal Pre-proof for 15 min at 1500xg and 4°C [15]. The resulting supernatants were extracted with equal volumes of ice-cold chloroform. Supernatants were then neutralized to a pH of 6 to 7 with 5 M NaOH solution and stored at -80°C until HPLC analysis. 2.6 Extraction of Mouse Plasma for HPLC Analysis Blood samples were collected into 1.5-ml Eppendorf micro-centrifuge tubes containing heparin (10,000 USP units/ml), nitrobenzylmercaptopurine ribonucleoside (NBMPR) (500 nM), and 2’-deoxycoformycin (500 nM) in 50 µl sterile distilled H2O [16].

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The tubes were gently mixed, and 600-800 μl of blood were immediately transferred to another set of 1.5-ml Eppendorf micro-centrifuge tubes on ice, followed by immediate Plasma was then removed and rapidly

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centrifugation at 15600xg for 2 min at 4°C.

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deproteinized with 70% w/w perchloric acid to reach a final perchloric acid concentration of 4%. Deproteinized samples were centrifuged at 15600xg for 2 min at 4°C, and the resulting

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supernatants removed and neutralized with 5 M NaOH to a pH of 6-7. The extracts were stored at -80°C until HPLC analysis. Numbers of individual mice tested in each category

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were as follows: WT, 21 (♀ 7, ♂ 14); mENT1 KO, 25 (♀ 13, ♂ 12); mENT2 KO, 34 (♀ 16,

2.7 HPLC

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♂ 18); mENT1/mENT2 double KO, 41 (♀ 22, ♂ 19); mCNT3 KO, 36 (♀ 22, ♂ 14).

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Analysis of purine and pyrimidine nucleosides and nucleobases was performed on an

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HPLC Agilent 1260 Infinity series system equipped with a quaternary pump with a built-in 4-channel degassing unit, an autosampler, a column temperature controller and a variable wavelength detector connected to an Agilent Open Lab running Chemstation software. Eleven nucleosides and nucleobases (adenosine, guanosine, inosine, uridine, cytidine, thymidine, adenine, thymine, hypoxanthine, uracil, and cytosine) were separated and analyzed using an Agilent Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm) and an Eclipse Plus C18 guard column (12.5 mm × 4.6 mm, 5 μm) at 22°C. The mobile phase was composed of water (A) and acetonitrile (B) with a gradient program as follows: 0-5 min, isocratic 1% B; 5-15 min, linear gradient 1-3% B; 15-25 min, linear gradient 3-6% B; 25-30 min, linear gradient 6-15% B; 30-35 min, linear gradient 15-1% B; 35-50 min, isocratic 1% B [17]. The flow rate was kept constant at 0.8 ml/min and the injection volume was either 15

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Journal Pre-proof μl (plasma extracts) or 30 μl (oocyte extracts). The variable wavelength detector was set at 254 nm.

Identification of nucleosides and nucleobases was determined by comparing

retention times and UV spectra against known standards.

Radioactivity detection was

performed on a Beta-RAM Model 5 radio HPLC detector equipped with a 500 μl cell volume radiochemical liquid cell at a scintillant flow rate of 1-2 ml/min. Fig. 3 shows a representative HPLC chromatogram for separation of nucleoside and nucleobase standards after a 20-min pre-equilibration time, resulting in retention times of 4.3 min (cytosine), 5.2 min (uracil), 6.4 min (cytidine), 7.2 min (hypoxanthine), 8.9 min (uridine), 10.9 min The limit of detection was ~ 0.1 μg/ml for each of the

nucleosides and nucleobases tested.

Plasma concentrations for WT and KO mice are

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and 29.2 min (adenosine).

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(thymine), 14.6 min (adenine) 18.0 min (inosine), 19.3 min (guanosine), 24.4 min (thymidine),

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presented as µM, and are means of triplicate determinations for each animal. Plasma extracts were prepared and analyzed in batches as animals became available from the breeding

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program.

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Section 2.8. Statistics

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P-values were determined by Student’s t-test.

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Journal Pre-proof 3. Results 3.1 HPLC Analysis of Nucleoside and Nucleobase Metabolism in Xenopus Oocytes Producing Recombinant Nucleoside Transporters The Xenopus laevis oocyte heterologous expression system was fundamental to the initial discovery and subsequent characterization of human CNT proteins because they lack, or have insignificant, endogenous nucleoside transport activity [13, 18-20]. The cDNAs encoding human and rodent ENTs and CNTs were all initially cloned and characterized in Xenopus oocytes [4, 6-9, 19, 22-28].

In the current work, we used oocytes producing

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recombinant hCNT3 to study how the metabolic fate of transported nucleosides in oocytes might potentially impact nucleoside transport measurements. We assessed the intracellular

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metabolism of transported radiolabelled [3H] and [14C] purine and pyrimidine nucleosides

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purine nucleosides at concentrations of 20 µM.

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after 1-min or 30-min incubation periods with medium containing individual pyrimidine or

Xenopus oocytes are reported to contain the following enzymes by which nucleosides

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and their corresponding nucleotides are metabolized: adenosine deaminase, nucleoside phosphorylase, cytidine deaminase, adenosine kinase, purine nucleoside phosphorylase,

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cytidine kinase, thymidine kinase, thymidine phosphorylase, nucleoside monophosphate kinase, and nucleoside diphosphate kinase. [29-32]. Some of the key metabolic steps are

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shown in Fig. 4A & B. Adenosine is converted to inosine by adenosine deaminase. Ribose from inosine and guanosine is removed, yielding ribose-1-phosphate and hypoxanthine and

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guanine, respectively, by purine nucleoside phosphorylase in reactions that are reversible. Salvage pathway rescue of hypoxanthine and guanine often occurs via hypoxanthine-guanine phosphoribosyltransferase. For pyrimidine nucleosides, cytidine is converted to uridine by cytidine deaminase.

Uridine and thymidine are then converted to uracil and thymine,

respectively, by pyrimidine nucleoside phosphorylase. Subsequent reactions convert uracil and thymine into β-alanine and β-aminoisobutyrate, respectively. Purine and pyrimidine nucleosides can also be phosphorylated by specific kinases.

Xenopus laevis oocytes

additionally possess endogenous ectonucleotidase activity [33]. Figs. 5A & B present representative HPLC chromatograms of radiochemical analysis of intracellular [3H]nucleosides (uridine, adenosine, cytidine, inosine, thymidine, or

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Journal Pre-proof guanosine) after 1-min or 30-min incubations with 20 µM [3H]nucleoside in hCNT3producing Xenopus oocytes. For uridine, after 1 min, 100% of radioactivity was uridine. After 30 min, 93% of radioactivity was uridine, and 3% was nucleotides (values calculated as percentages of the total radioactivity detected in the corresponding samples). For adenosine, after 1 min, 100% of radioactivity was adenosine. After 30 min, 49% was adenosine, 38% was nucleotides, 8% was adenine and 5% was inosine. For inosine, after 1 min, 100% of radioactivity was inosine. After 30 min, 84% was inosine, 9% was nucleotides and 6% was hypoxanthine.

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Cytidine, thymidine and guanosine were unchanged after both 1 and 30 min.

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Thus, nucleosides transported by hCNT3 in oocytes after 1-min incubations were

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not subject to significant intracellular metabolism, and after 30-min incubations were generally subject to minimal intracellular metabolism (uridine 7%, cytidine 0%, thymidine

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0%, inosine 16% and guanosine 0%), with the exception of adenosine, for which 49%

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remained unmetabolized.

3.2. HPLC Analysis of Adenosine Interactions with Recombinant hCNT1 Produced in Xenopus Oocytes

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Previously, it was found that rCNT1 mediates efflux of [3H]uridine from preloaded oocytes, demonstrating a low, but significant, capacity for bidirectional transport of Uridine efflux was stimulated by extracellular uridine, but

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nucleoside permeants [4].

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inhibited by extracellular adenosine, suggesting that the rate of conversion of rCNT1 from outward-facing to inward-facing conformation was (i) increased when complexed with uridine, and (ii) decreased when complexed with adenosine. Thus, although rCNT1 binds adenosine and uridine with similar apparent affinities, subsequent transport kinetically favors uridine [4]. A small inward current in response to adenosine has also been observed for hCNT1 produced in oocytes [34], but there is some disagreement in the literature whether adenosine can be considered an hCNT1 permeant [35]. In the hCNT1 electrophysiological experiments of Fig. 6, we demonstrate and confirm that adenosine (100 µM) does indeed induce a small, but significant inward current in hCNT1-producing oocytes, that this current requires Na+, and that current was absent from control water–injected oocytes. Fig. 6 further demonstrates that the hCNT1 inward Na+ current induced by 100 µM uridine is very

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Journal Pre-proof much reduced when adenosine (100 µM) is also present. As further verification that adenosine functions as a low Km, low Vmax permeant of hCNT1, we then undertook hCNT1 [3H]uridine efflux experiments complementary to those previously reported for rCNT1 [4], but taking the important additional step of using HPLC to confirm the molecular identity of effluxed radioactivity. To do this, oocytes producing hCNT1 were preloaded with 10 µM [3H]uridine, after which the time course of efflux of intracellular radioactivity was measured over 60 min in transport medium alone, or in the presence of extracellular non-radiolabelled adenosine or uridine. As shown in Fig. 7, and

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similar to rCNT1, efflux of intracellular radioactivity against the inwardly-directed Na+

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electrochemical gradient in transport medium alone was relatively slow (23% decrease in intracellular radioactivity over 60 min), whereas the addition of 1 mM non-radiolabelled

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uridine to the extracellular medium resulted in a large stimulation of efflux (63% decrease in intracellular radioactivity over 60 min). In marked contrast to the trans-stimulation seen with

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uridine, 1 mM extracellular adenosine caused a substantial trans-inhibition of efflux (9%

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decrease in intracellular radioactivity over 60 min).

We then used radiochemical HPLC analysis to determine what proportion of

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radioactivity in both extracellular and intracellular fluids was present as [3H]uridine after 60 min of efflux. Although intracellular radioactivity was distributed between uridine (34 -

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77%) and nucleotides (23 – 66%), depending on conditions, almost all (97%) of the radioactivity exiting cells when stimulated by extracellular non-radiolabelled uridine was

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unmetabolized radiolabelled uridine (Tables 1 & 2), consistent with hCNT1-mediated efflux of uridine. Likewise, unmetabolized radiolabelled uridine was the major efflux product in oocytes incubated in the absence of extracellular nucleoside, or in the presence of extracellular non-radioactive adenosine. 3.3. HPLC Analysis of the Roles of Individual ENTs and CNTs in Nucleoside and Nucleobase Homeostasis in Mice Owing to the overlapping specificities, distributions and other characteristics of NT proteins, the physiological importance and roles of individual NTs have been difficult to assess. Mice (m) possess mouse homologues of human ENTs and CNTs, and the functional

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Journal Pre-proof characteristics of individual mENTs and mCNTs resemble those of their human counterparts [1, 6, 7, 25, 36-38]. Therefore, generation of mice with targeted disruptions of the genes encoding various NT proteins offers a valuable tool in gaining understanding of roles and importance of individual transporters in transport and homeostasis of various nucleosides and nucleobases. For the present series of experiments, mENT1 and mENT2 homozygous (-/-) knockout (KO) mice were produced from heterozygous (+/-) mice as described previously for mENT1 [14, 39]. In turn, mENT1 homozygous KO mice were crossed with mENT2

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homozygous KO mice to generate a mENT1/mENT2 double KO knockout strain. KO

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mice deficient in mCNT3 were also produced, allowing us to examine changes in plasma concentrations of nucleosides and nucleobases in response to individual and combined

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deletions of mENT1/2 in comparison to those in response to deletion of mCNT3. Mice

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deficient in mCNT3 have not been described previously.

Plasma samples from WT control and KO mice were collected and analyzed by the

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HPLC Agilent 1260 infinity series system as described in Materials and Methods. Concentrations of the following nucleosides and nucleobases were quantified: adenosine,

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uridine, thymidine, cytidine, guanosine, cytosine, uracil, hypoxanthine, thymine and adenine.

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For purine nucleosides (Table 3), the concentration of adenosine in plasma of WT mice was 0.9 + 0.1 µM. Compared to WT mice, adenosine plasma levels were increased

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12.3-fold in mENT1 KO mice, 2.3-fold in mENT2 KO mice, 2.2-fold in mCNT3 KO mice, and 10.4-fold in mENT1/mENT2 double KO mice. The concentrations of guanosine in all plasma samples were below the limit of detection (< 0.1 µM). The concentration of inosine in plasma of WT mice was 0.9 + 0.1 µM, and was increased by 2.4-fold in mENT1 KO mice, 1.4-fold in mENT2 KO mice, and 1.4-fold in mENT1/mENT2 double KO mice. Inosine concentrations in the plasma of mCNT3 KO mice were similar to those in WT mice. For pyrimidine nucleosides (Table 3), the concentration of uridine in plasma of WT mice was 10.0 + 0.1 µM, and was increased 1.4-fold in each of mENT1 KO mice, mENT2 KO mice and mENT1/mENT2 double KO mice. Its concentration in plasma of mCNT3

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Journal Pre-proof KO mice was similar to that of WT mice. The concentration of cytidine in plasma of WT mice was 6.6 + 1.0 µM, and was increased 1.4-fold in mENT2 KO mice, and 2.4-fold in mENT1/mENT2 double KO mice. Cytidine concentrations in the plasma of mENT2 KO and mCNT3 KO mice were not significantly different from WT mice. The concentration of thymidine in plasma of WT mice was 0.4+ 0.1 µM, and was increased by 1.8-fold in mENT1 KO mice, 1.3-fold in mENT2 KO mice, and 2.8-fold in mENT1/mENT2 double KO mice. Thymidine concentrations in the plasma of mCNT3 KO mice were similar to those in WT mice.

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For nucleobases (Table 3), the concentration of uracil in plasma of WT mice was

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10.3 + 1.0 µM, and was reduced by 30% in mENT2 KO mice, by 34% in mENT1/mENT2 double KO mice, and by 37% in mCNT3 KO mice. Uracil concentrations in the plasma of

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mCNT3 KO mice were not significantly different from those in WT mice.

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concentration of adenine in plasma of WT mice was 2.3 + 0.1 µM, and was increased by 1.2

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to 1.5-fold in each of the KO strains. The concentrations of thymine in all plasma samples

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were below the limit of detection (< 0.1 µM). The concentration of hypoxanthine in plasma of WT mice was 10.4 + 0.4 µM, was similar to WT in both mENT2 KO mice and mCNT3 double KO mice.

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KO mice, and was reduced by 21% in mENT1 KO mice, and by 38% in mENT1/mENT2

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Since there is evidence of possible gender differences in ENT1-related behaviours or responses in mice [40], the plasma nucleoside and nucleobase values in Table 3 were also

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analyzed separately for female and male mice. Disaggregated data for plasma nucleoside and nucleobase concentrations were generally similar for females and males, with the notable exception of adenosine. Fig. 9 shows the distributions of plasma adenosine concentrations in female and male WT and KO mice, revealing that elevations in plasma adenosine concentrations for mENT1 KO mice and mENT1/mENT2 double KO mice occurred in both sexes, whereas increases in plasma adenosine concentrations for both mENT2 KO mice and mCNT3 KO mice only occurred in females. In females, mean plasma adenosine values (+ SEM (n)) in WT mice, mENT1 KO mice, mENT2 KO mice, mENT1/mENT2 double KO mice and mCNT3 KO mice were 1.2 + 0.3 (7), 12.3 + 0.5*¶ (13), 3.9 + 0.3*#¶ (16), 10.6 + 0.3*#¶ (22) and 4.3 + 0.3*#¶ (22) µM, respectively, where symbols *, #, and ¶ indicate a significant difference [P < 0.05] compared to either female WT mice, female 15

Journal Pre-proof mENT1 KO mice, or male KO mice of the same strain, respectively. Corresponding plasma adenosine concentrations in male mice were 0.8 + 0.1 (14), 9.9 + 0.5* (12), 0.6 + 0.4* (18), 8.2 + 0.3*# (19) and 0.4 + 0.1*# (14) µM, respectively, where symbols * and # indicate a significant difference [P < 0.05] compared to either male WT mice, or male mENT1 KO mice. Consistent with the plasma adenosine gender difference in mENT2 KO mice, the plasma adenosine concentrations in mENT1/mENT2 double KO mice were lower [P < 0.05] in males compared with females (8.2 + 0.3 (19) µM versus 10.6 + 0.3 (22) µM,

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respectively).

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Journal Pre-proof 4. Discussion In the present paper, we have employed the technique of HPLC to address unresolved issues in nucleoside membrane transport and physiology. The Xenopus oocyte heterologous expression system is widely used in functional studies of recombinant human and other NTs, but the extent to which accumulated nucleosides are subject to intracellular metabolism is largely unknown. In the present study, we have extended a previous thin layer chromatography analysis of uridine [41] to

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demonstrate that hCNT3-transported uridine and other radiolabelled purine and pyrimidine nucleosides in Xenopus laevis oocytes after 1 min incubation were not subject to significant

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metabolism. After 30 min, there was detectable metabolism of uridine (7%) and inosine (16%), and substantial metabolism of adenosine (51%), but little, if any, metabolism of In most nucleoside transport studies using Xenopus

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cytidine, thymidine or guanosine.

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oocytes as the heterologous expression system, initial rates of transport are determined using 1-min and 5-min uptake intervals for CNTs and ENTs, respectively. Thus, there is minimal

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nucleoside metabolism within the incubation periods typically used in initial rate determinations for both transporter families. Together with low endogenous nucleoside

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transport activity, therefore, Xenopus oocytes have the additional advantage as a heterologous expression system of low (except for adenosine) intracellular nucleoside metabolism. Even in

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the case of adenosine, the dominant metabolic fate was phosphorylation into impermeable derivatives, and thus would not interfere with determination of initial rates of transport. The

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adenosine deaminase inhibitor 2’-deoxycoformycin is often used in radioisotope flux assays with adenosine to block possible conversion to inosine and then hypoxanthine (e.g. [42]). 2’Deoxycoformycin was also included in the experiments reported here. After 30 min of uptake, only 5% of intracellular adenosine had been converted to inosine, and no intracellular hypoxanthine was detected, suggesting that 2’-deoxycoformycin had fulfilled its role. These findings are reported in the context of using the Xenopus oocyte heterologous expression system for functional and mechanistic studies of recombinant hNTs, and not because we consider Xenopus oocytes a physiological model for the interplay between nucleoside transport and subsequent intracellular metabolism.

17

Journal Pre-proof Similar to previous transport studies with rCNT1, we confirmed that adenosine is an hCNT1 permeant, and also established that recombinant hCNT1-mediated efflux of [3H]uridine from preloaded oocytes was stimulated by extracellular uridine and inhibited by extracellular adenosine, with > 95% of the radioactivity exiting cells being unmetabolized radiolabelled uridine. The interpretation of this and other findings for rCNT1 was that adenosine functions as a low Km, low Vmax permeant, the transporter complexed with adenosine reorienting its permeant binding site from out-to-in more slowly than when unoccupied, or when complexed with uridine (mobility of uridine-complexed carrier > empty carrier >

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adenosine-complexed carrier). The validity of this interpretation rests on the assumption that effluxed radioactivity measured in the experiment is still in the form of uridine. HPLC analysis

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of extracellular fluids in the efflux experiments reported here with hCNT1-producing

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oocytes established this to be the case. Interaction of adenosine with hCNT1 allows this nucleoside to act, in appropriate circumstances, as an hCNT1 inhibitor, providing a potential

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means of purine nucleoside regulation of a pyrimidine nucleoside-selective transporter. It remains to be established whether there are physiological or pathological conditions under

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which adenosine concentrations rise sufficiently to elicit such inhibition.

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We regard discrepancies in the literature as to whether adenosine is indeed a CNT1 permeant as technical in nature. In [35], for example, current recordings were relatively unstable, making very small currents of 1-2 nA difficult to measure. In our experiments,

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currents always returned to baseline upon removal of adenosine or other permeants. Unlike

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[35], we also included 2’-deoxycoformycin in perfusions to block adenosine deaminase activity, thereby minimizing degradation of adenosine during the experiment. The prototypic equilibrative nucleoside transporter hENT1 has long been recognized as a potential target for therapeutic strategies based on modulation of transport of physiological nucleosides and anticancer nucleoside drugs [1, 43]. Especially important is the control of adenosine levels at extracellular surfaces, and the consequent influence on purinoreceptor intracellular signaling cascades. Like hENT1, hENT2 exhibits broad permeant selectivity, but is much less well understood than hENT1, largely because most cell types co-express both transporters, with hENT1 typically showing the greater activity. In addition to nucleosides, both transporters also transport nucleobases [42].

hCNT3 also exhibits broad permeant

selectivity for purine and pyrimidine nucleosides but, unlike hENT1 and hENT2, does not 18

Journal Pre-proof transport nucleobases. Mice possess NTs homologous to those in humans, and the generation of KO mice with targeted disruptions of the genes encoding the various mouse transporters offers a means for in vivo investigation of their roles in nucleoside physiology and homeostasis. In the present study, we used HPLC to investigate plasma concentrations of nucleosides and nucleobases in mice with disruptions of the genes for mENT1, mENT2, or mCNT3 or for both mENT1 and mENT2. The objective was to test if deficiencies in the three broad selectivity transporters resulted in plasma concentration phenotypes indicative of roles in plasma nucleoside/nucleobase homeostasis.

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The plasma concentrations of nucleosides and nucleobases detected in WT mice

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were similar to values reported in the literature [44-46]. The results for KO mice provide clear and strong evidence for a major role of mENT1 in adenosine homeostasis, with

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secondary, possibly female-specific, contributions to adenosine homeostasis from mENT2 and mCNT3. Contributions of mENT1 to inosine uridine and thymidine homeostasis were

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also apparent. None of the transporters were major participants in handling of nucleobases.

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Studies of mENT1 KO mice reveal that mENT1 plays an important role in ethanol preference and consumption [47], anxiety-related behavior [48], cardioprotection during

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ischemia [49, 50], soft tissues calcification [51], sleep-wake regulation [52], response to caffeine [53], and altitude adaptation [54]. These diverse changes can be explained by altered

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ENT1-mediated control of adenosine levels and adenosine receptor signaling, the presently reported large increase in plasma adenosine concentration in mENT1 KO mice being

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substantially greater than more modest (~ 2-fold) elevations reported previously [50, 51], or the ~ 50% decrease seen in mENT2 KO dialysates of the mouse forebrain nucleus accumbens [55], possibly reflecting the care we took to minimize adenosine uptake into erythrocytes and metabolism during sample preparation. Specifically, blood samples were collected in the presence of the ENT1 inhibitor NBMPR and the adenosine deaminase inhibitor 2’-deoxycoformycin, and all steps were performed at 4 ºC.

Furthermore, by

analyzing the full spectrum of changes in plasma nucleoside and nucleobase levels in mENT1 KO mice, our data reveal and contrast the unique physiological importance of ENT1 in adenosine homeostasis versus that for other nucleosides. Changes in plasma adenosine levels in response to deletion of mENT2 or mCNT3 were much more modest, and are consistent with an elevation of adenosine in mENT2 KO bronchoalveolar fluid [56]. 19

Journal Pre-proof A potentially important finding was that the elevation of plasma adenosine concentrations in mENT2 KO mice and mCNT3 KO mice was gender-specific, the response only occurring in females. This indicates a potential complexity in the roles of NTs in plasma adenosine regulation not previously appreciated. For other nucleosides and nucleobases, deletion of mENT1, mENT2 or mCNT3 had only modest effects. The recently solved 3D crystal structures of ENT and CNT family members in different conformational states and with different bound nucleosides or inhibitors [10-12] has advanced our understanding of nucleoside transport at the molecular level, revealing the

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identities of individual amino acid residues within the permeant binding pockets, and

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demonstrating the different and contrasting conformational transitions by which nucleoside translocation occurs. Adenosine is an atypical CNT1 permeant, binding adenosine with high

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affinity (low Km) similar to other nucleosides, but with a very much reduced translocation capacity (Vmax) that is attributed to a uniquely reduced transmembrane mobility of the

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adenosine-carrier complex. Identification of the molecular basis of this reduced mobility will

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be both challenging and informative, and requires sequence and mutagenesis comparisons

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with, for example, CNT3, which transports adenosine “normally”.

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Journal Pre-proof Figure legends Figure 1. Schematic representation of modified mouse ENT and CNT genes. Figure 2.

PCR genotyping of mCNT3 KO mice. (A) The deletion site of a gene-trap

vector of the slc28a3 (mCNT3) gene is shown.

Also indicated are the locations and

sequences of the PCR primers used for genotyping, and sizes of each of the PCR products generated. (B) Representative agarose gel electrophoresis of PCR products obtained for

Figure 3.

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individual CNT3 (+/+), CNT3 (+/-) and CNT3 (-/-) mice. Representative HPLC chromatogram of nucleoside and nucleobase

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standards. The y-axis is UV absorbance in milli-Absorbance Units (mAU), and the x-axis is

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retention time (in min). Please see Materials and Methods for experimental details. Figure 4. Pathways for nucleoside catabolism. (A) purine nucleosides, (B) pyrimidine

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nucleosides.

producing Xenopus oocytes.

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Figure 5. Radiochemical HPLC analysis of intracellular [3H] nucleosides in hCNT3Shown are representative HPLC chromatograms of

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nucleoside- or nucleobase-associated intracellular radioactivity in oocytes after 1-min (A) or 30-min (B) incubation periods with individual [3H]/[14C]nucleosides or nucleobases (as

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indicated) present extracellularly at 20 µM. Values are for groups of 20 oocytes. The y-axes

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represent quantities (dpm) and the x-axes represent is retention times (min). Figure 6. Electrophysiological demonstration of adenosine as a low-capacity hCNT1 permeant. Steady-state currents induced by 100 µM uridine, 100 µM adenosine, or 100 µM each of uridine and adenosine in Xenopus oocytes producing recombinant hCNT1 in the presence or absence of Na+ are presented as means (+ SEM) for 4-6 different oocytes. No currents were detected in control water-injected oocytes (data not shown). Figure 7. Trans-stimulation and trans-inhibition of hCNT1-mediated uridine efflux, respectively, by extracellular uridine and adenosine.

Efflux of radioactivity was

measured from hCNT1-producing oocytes preloaded with 10 μM [3H]uridine for 30 min at 20°C suspended in NaCl transport medium alone (●) or in NaCl transport medium

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Journal Pre-proof containing 1 mM non-radioactive uridine (○) or 1 mM non-radioactive adenosine (▼). Each data point represents efflux from a group of 20 oocytes. Figure 8. Radiochemical HPLC analysis of extracellular and intracellular radioactivity in hCNT1-producing oocytes. Groups of 20 hCNT1-producing oocytes were pre-loaded with 20 µM [3H]uridine for 30 min, washed, and resuspended in medium with or without 1 mM adenosine or uridine. After 60 min, HPLC analysis of extracellular and intracellular radioactivity associated with uridine and nucleotides was conducted as

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described in Materials and Methods. Figure 9. Plasma adenosine levels measured by HPLC in female and male wild-type,

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mENT1 KO, mENT2 KO, mENT1/mENT2 double KO or mCNT3 KO mice.

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Values for individual female (solid squares) and male (open squares) mice are from Table 3.

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Please see text for means + SEM (n).

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Journal Pre-proof Table 1. Summary of radiochemical HPLC analysis of extracellular radioactivity in hCNT1-producing oocytes. Sample volumes were 30 µl, and values are means + SEM (n = 3) following 60-min efflux from groups of 20 [3H]uridine-containing oocytes either in the absence or in the presence of extracellular nonradioactive uridine or adenosine. Please see

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Materials and Methods for experimental details.

Uridine

NaCl

Radioactivity (dpm)

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Extracellular

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Efflux condition

3958 ± 151

93

320 ± 33

7

Uridine

12068 ± 200

97

Nucleotides

388 ± 36

3

Uridine

1200 ± 68

74

412 ± 55

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Nucleotides

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Nucleotides

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1 mM adenosine

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1 mM uridine

% of total radioactivity

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Journal Pre-proof Table 2. Summary of radiochemical HPLC analysis of intracellular radioactivity in hCNT1-producing oocytes. Sample volumes were 30 µl, and values are means + SEM (n = 3) following 60-min efflux from groups of 20 [3H]uridine-containing oocytes either in the absence or in the presence of extracellular nonradioactive uridine or adenosine. Please see

% of total radioactivity

5300 ± 179

77

Nucleotides

1562 ± 70

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Uridine

496 ± 45

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Nucleotides

954 ± 101

66

Uridine

6446 ± 176

76

Nucleotides

2034 ± 125

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Uridine

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NaCl

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1 mM uridine

1 mM adenosine

Radioactivity (dpm)

Intracellular

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Efflux condition

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Materials and Methods for experimental details.

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Journal Pre-proof

mENT1 KO

mENT2 KO

mENT1/mENT2 double KO

mCNT3 KO

Number of mice

21

25

34

41

36

Adenosine (µM)

0.9 ± 0.1 (100)

11.1 ± 0.4* (1230)

2.1 ± 0.2* (233)

9.4 ± 0.3*# (1040)

2.1 ± 0.2* (224)

Guanosine (µM)

0.0 (nd)

0.0 (nd)

0.0 (nd)

0.0 (nd)

0.0 (nd)

Inosine (µM)

0.9 ± 0.1 (100)

2.2 ± 0.2* (244)

1.3 ± 0.1* (144)

0.8 ± 0.1 (89)

Uridine (µM)

10.0 ± 0.5 (100)

14.3 ± 0.5* (143)

13.5 ± 0.4* (135)

14.2 ± 0.5* (142)

10.2 ± 0.3 (103)

Cytidine (µM)

6.6 ± 1.0 (100)

9.2 ± 0.6* (140)

9.8 ± 0.9 (149)

15.6 ± 0.8* (236)

5.1 ± 0.5 (77)

Thymidine (µM)

0.4 ± 0.1 (100)

0.7 ± 0.1* (175)

0.5 ± 0.03* (125)

1.1 ±0.02* (275)

0.4 ± 0.02 (107)

Uracil (µM)

10.3 ± 1.0 (100)

8.1 ± 0.7 (79)

7.2 ± 0.5* (70)

6.8 ± 0.4* (66)

6.5 ± 0.6* (63)

Adenine (µM)

2.3 ± 0.1 (100)

2.9 ± 0.1* (126)

2.9 ± 0.1* (126)

3.5 ± 0.1* (152)

2.9 ± 0.1* (122)

0.0 (nd)

0.0 (nd)

0.0 (nd)

0.0 (nd)

0.0 (nd)

10.4 ± 0.4 (100)

8.2 ± 0.5* (79)

12.9 ± 0.6 (124)

6.4 ± 0.2* (62)

10.2 ± 0.4 (98)

Hypoxanthine (µM)

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1.3 ± 0.1* (144)

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Thymine (µM)

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Wild-type

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Table 3. Plasma nucleosides and nucleobases levels measured by HPLC in wildtype, mENT1 KO, mENT2 KO, mENT1/mENT2 double KO or mCNT3 KO mice. Please see Materials and Methods for experimental details. Values are mean concentrations (µM) + SEM of the number of mice indicated in the table, and the % of control is presented in parenthesis. Distributions of adenosine concentrations in female and male mice are presented in Fig. 9.

* P < 0.05 (compared to WT) # P < 0.05 (compared to mENT1 KO) nd: not detected

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Journal Pre-proof Acknowledgement

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This work was supported in part by the Canadian Institutes of Health Research (grant project number: G118160061).

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Journal Pre-proof Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Graphical abstract Highlights ● HPLC was used to study nucleoside transport in Xenopus oocytes and knockout mice ● Oocytes subjected transported nucleosides to minimal intracellular metabolism ● Adenosine was an atypical permeant of concentrative nucleoside transporter CNT1 ● Equilibrative nucleoside transporter ENT1 determined plasma adenosine levels

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