BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 233, 572–575 (1997)ARTICLE NO. RC976498

Expression of Sodium-Dependent Purine NucleosideCarrier (SPNT) mRNA Correlates with NucleosideTransport Activity in Rat Liver

Antonio Felipe, Andreu Ferrer-Martınez, F. Javier Casado, and Marcal Pastor-AngladaDepartament de Bioquımica i Biologia Molecular, Universitat de Barcelona, Diagonal 645, 08071 Barcelona, Spain

Received March 19, 1997

with the idea of stable changes in the number of carri-The expression of sodium-dependent purine nucleo- ers in the hepatocyte membrane (4). Glucagon was able

side transport (SPNT) mRNA has been studied in phys- to upregulate the sodium-dependent nucleoside trans-iological situations in which Na/-dependent nucleo- port in hepatocytes in short-term. This effect was tran-side uptake in plasma membrane vesicles from rat sient and dependent on membrane potential, and corre-liver was induced. Sodium-dependent uridine trans- lated with the electrogenic nature of the uptake (4). Inport rates were induced in genetically obese Zucker those situations described above, Na/,K/ ATPase alsorats, during liver regeneration after partial hepatec- up-regulated the activity and this induction was corre-tomy, and under euglycemic-hyperinsulinemic clamp. lated with an increase in the mRNA and protein ex-A PCR-generated fragment, based on a published

pression levels of the a1 subunit of Na/, K/ ATPaseSPNT sequence cloned from rat liver, was used as a(6,7,8).probe in Northern blot analysis. We show that the he-

At least three sodium-dependent nucleoside trans-patic mRNA levels of the putative sodium-dependentport systems have been described kinetically in epithe-transport system SPNT correlate with the sodium-de-lia, N1, N2 and N3 (9). N1 is a purine-preferring sys-pendent uridine transport rates in plasma membranetem, N2 has been described as pyrimidine-preferringvesicles from rat liver. These results suggest that theand N3 accepts a broad range of substrates. Recently,induction of the sodium-dependent nucleoside trans-the putative cDNAs encoding N1- and N2-related pro-port expressed in liver parenchymal cells involves reg-teins have been isolated. cNT1, a cDNA encoding aulation of SPNT gene expression. q 1997 Academic Presspyrimidine-preferring nucleoside transport system (N2),was found to be present in kidney and jejunum (10).SPNT, a cDNA cloned from rat liver and also presentin rat jejunum, leads to the induction of a sodium-de-Sodium-dependent nucleoside transport mainly oc-pendent purine-preferring nucleoside transport activ-curs in epithelial tissues, such as kidney and intestine,ity when expressed in Xenopus oocytes (11).although it has also been found in several cultured cell

In order to study to what extent the regulation of thelines (1,2). Liver cells also present sodium-dependenthepatic sodium-dependent nucleoside transport activ-nucleoside transport similar to that described in cellsity relies upon changes in the mRNA amounts of theof epithelial origin (3). This transport activity is undernucleoside transporter isoform gene, we determinedcomplex regulation and it is induced in several situa-whether the changes in the transport activity corre-tions in both short and long-term (4). In the early phaselated with a differential expression of the SPNT mRNAof liver growth after partial hepatectomy (liver hyper-levels in rat liver. Here we show, for the first time, thatplasia) and in genetically obese Zucker rats (liver hy-SPNT mRNA levels increase in the same situations inpertrophia), Na/-dependent uridine transport is in-which an induction of the sodium dependent nucleosideduced in plasma membrane vesicles from liver. Thistransport activity has been described. The present re-induction remained stable after monensin treatmentport suggests that the expression of the sodium-depen-and led to an increase in the Vmax of the transportdent nucleoside transport system in liver parenchymalwithout changes in the Km. These findings are consis-cells involves either transcriptional or pre-tanslationaltent with an increase in the number of carriers at thesteps, or both.plasma membrane level rather than a change in Na/

permeability (5,6). A similar induction has been found MATERIALS AND METHODSin livers from animals kept under euglycemic-hyperin- Animals and experimental groups. Partial hepatectomy was per-

formed in overnight-fasted male Wistar rats (200-240 g b. wt.), assulinemic clamp for 6 hours. This is also compatible

0006-291X/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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described in (12). Briefly, after pentobarbital anaesthesia (60 mg/ TABLE 1kg b.wt. i.p.) rats were laparatomized and a partial hepatectomy of Uridine Transport in Plasma Membrane Vesiclesaproximately 70% was performed. Sham-operated rats used as con-

from Rat Livertrol did not undergo hepatectomy but only a liver extrusion. At theindicated times, 6 h for transport studies and 2 h for RNA isolation,

Partial hepatectomyanimals were sacrified.Euglycemic-hyperinsulinemic clamp was done as described else-

Sham Hepatectomizedwhere (13). In summary, male Wistar rats (200-240 g b.wt.) wereused for surgery under pentobarbital anaesthesia as described above.

Na/ 0.190 { 0.020 0.270 { 0.020*Animals showed a full recovery a few hours after surgery. The experi-K/ 0.130 { 0.010 0.120 { 0.010ments were performed in overnight-fasted animals 24 hours afterNa/-dependent 0.060 { 0.005 0.150 { 0.010***the implantation of catheters. The euglycemic-hyperinsulinemic

clamp was performed as previously described (13). Animals were Euglycemic-hyperinsulinemic clampkilled 6 hours after the initiation of the clamp. Saline-infused ani-mals were used as controls. Control Clamp

Six-week-old male Zucker rats were used for the experiments. Thebody weight for genetically obese animals and their lean littermates Na/ 0.531 { 0.026 0.665 { 0.031*was around 245 g and 180 g respectively. These values allowed us K/ 0.423 { 0.036 0.482 { 0.037to determine two different animal populations as we previously de- Na/-dependent 0.108 { 0.005 0.182 { 0.010***fined in (14).

All animals were killed by decapitation and liver samples were Genetically obese Zucker ratimmediately either processed for plasma membrane vesicle prepara-tion or frozen in liquid nitrogen and kept at 0807C for total RNA Lean Obeseextraction.

Na/ 0.495 { 0.041 1.140 { 0.060***Preparation of plasma membrane vesicles from rat liver. AfterK/ 0.230 { 0.050 0.350 { 0.030liver excision, samples were used for plasma membrane isolationNa/-dependent 0.270 { 0.057 0.790 { 0.024***following (15). This method, widely used and validated in our labora-

tory, involves a Percoll density gradient by high-speed centrifugationNote. Uridine (1mM) transport was determined as described inand yields a considerable amount of plasma membrane with low

Materials and Methods. Transport rates are expressed in pmol ofcontamination by other subcellular membranes. Vesicle preparationsuridine/mg prot/3s. Values are means{ SEM of at least four indepen-from regenerating rat livers were obtained after pooling the liversdent plasma membrane preparations each performed in triplicate.from two to three animals. The plasma membrane preparation re-Statistical differences between the preparations from hepatectom-tains most transport capacities as previously described (5,6,13,14).ized (6h), clamp (6h), and obese rats and their respective counter-The protein content of the preparations was measured according toparts were compared by Student’s t test (*Põ0.05; ***Põ0.001).Bradford (16).

Transport measurements. Uridine uptake measurements wereperformed by a filtration method adapted from (17) as describedpreviously (3). The advantages for using uridine as substrate as well 7,13,14), and only those fractions that retained concen-as the specific conditions for transport studies are described in (3). trative sodium-dependent alanine transport activityThe experiments were carried out at 3s (near initial velocity condi-

were used (not shown). Table 1 shows that, in all thetions), in the presence of a transmembrane gradient of 0.1M NaSCNor 0.1M KSCN and 1mM [3H] uridine as substrate at 227C. All the physiological situations analyzed in this study, uridineexperiments were performed in triplicate in at least four independent transport activity is increased over their control coun-vesicle preparations. terparts. This induction involves exclusively the Na/-

RNA extraction and Northern blot analysis. Liver tissue was ho- dependent component of transport because uridine up-mogenized in a solution containing 4M guanidine thiocyanate, 25take in a KSCN medium is unchanged. Partial hepatec-mM sodium citrate, 0.5% sarkosyl, and 100mM 2-mercaptoethanol.tomy causes a 2.5 fold increase in the concentrativeTotal RNA was extracted by the single step method of Chomcynski

and Sacchi (18). RNA (20mg) was fractionated by electrophoresis uridine transport capacity in the early phase of liverthrough a 1% agarose-3% formaldehyde gel in 20mM 3-(N-morpho- growth (six hours after surgery), when compared withlino) propanesulfonic acid and 1 mM EDTA, pH 7.4. Application of the sham-operated controls. Similarly, a six hours eu-equal amounts of RNA to each lane was confirmed by the addition

glycemic-hyperinsulinemic clamp significantly up-reg-of ethidium bromide to the samples before electrophoresis. The gelulates the Na/-dependent uridine transport activitytreatment and the high stringency hybridrization conditions are de-

scribed elsewhere(19). Filters were hybridized with 106 cpm/ml of (70% over the saline infused animals). Obese Zuckerthe PCR-generated cDNA SPNT fragment (nucleotides 675 to 1465) rats showed a three fold increase in sodium-coupledfrom the published sequence (11). The fragment was amplified from nucleoside transport in comparison to their lean litterrat liver cDNA, blunted with Klenow fragment and ligated into the

mates.Eco RV site from BlueScript KS.The SPNT cDNA sequence was con-firmed using the Auto Read sequencing kit and the A.L.F. DNA The Northern blot analysis of SPNT and 18S in theSequencer (Pharmacia). A further hybridization with a 200 bp cDNA experimental groups are depicted in figure 1. The re-probe to 18S ribosomal RNA was used as control. The Northern blot sults show that mRNA levels for the sodium-dependentanalysis was performed in two different filters with three indepen-

purine preferring nucleoside transporter (SPNT) in-dent RNA samples in each one.creased in the three experimental situations. No

RESULTS AND DISCUSSION changes were observed in the amounts of 18S RNA,which is routinely used as a loading and transferringThe plasma membrane preparations used in this

study were characterized, as previously described (5- control. This finding supports the view that, in these

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The early stages of genetic obesity are characterizedby liver hypertrophy and hyperinsulinemia (21). Livergrowth associated to the development of the obese phe-notype in Zucker rats may have little in common withliver regeneration, but the induction in the Na/-depen-dent nucleoside transport activity and its correspond-ing increase in SPNT mRNA amounts, is probably theresult of the established hyperinsulinemia. Insulin isable to induce Na/-dependent nucleoside transport ac-tivity in primary cultures of rat hepatocytes (4). Thistype of regulation can be also found using in vivo mod-els, such as the euglycemic-hyperinsulinemic clamp.This study shows that insulin plays a key role in theregulation of hepatic sodium-dependent nucleoside

FIG. 1. Northern blot analysis of sodium-dependent purine pre- transporter expression. Nevertheless, since it is knownferring nucleoside transport (SPNT) in rat liver. A representativethat insulin by itself may be either a mitogenic factorNorthern blot is shown. Up to 20mg of liver total RNA was electropho-or a potentiating agent of proliferation in some culturedresed as described in Materials and Methods. Upper panel, SPNT

specific hybridization—a blot segment showing the Ç2.9 Kb tran- cell lines and hepatocytes (22) it is interesting to high-script. Lower panel, 18S ribosomal specific hybridization—a seg- light the possibility that insulin plays a role in SPNTment showing the Ç1.8 Kb 18S transcript. S, sham-operated; R, induction in proliferative conditions such as liver re-2h hepatectomized; C, saline infused control rats; H, euglycemic-

generation after partial hepatectomy.hyperinsulinemic clamp; L, lean Zucker; O, obese Zucker.In summary, in the present work we have shown

that an induction in the sodium-dependent nucleosidetransport in liver correlates with a greater abundancephysiological conditions, the increase in Na/-coupled

uridine transport is the consequence of a stable change in SPNT mRNA levels and suggests that hyperinsulin-emia and growth-associated states may associated withat the plasma membrane level that is accompanied by

enhanced SPNT mRNA levels, thus paralleling what transcriptional and/or pre-translational regulation ofthe SPNT gene.happens with the Na/,K/-ATPase. The activity of the

pump is simultaneously induced in these models in away which depends on an increase in the a1 and b1 ACKNOWLEDGMENTSsubunit mRNA and protein amounts (6,7,8). Here weshow, for the first time, that an induction on the nucleo- This work has been supported by Grants PB92-0867 and PB95-side transport rates, that appears to be consistent with 0975 from DGICYT (Ministerio de Educacion y Ciencia) and Marato

TV3 (Fundacio August Pi i Sunyer). The pBR18S plasmid was kindlyde novo synthesis of carrier proteins, relies upon tran-provided by Dr. Isabel Fabregat (Departamento de Bioquımica y Bio-scriptional or pre-translational regulation of the re-logıa Molecular, Universidad Complutense de Madrid).cently cloned SPNT carrier gene.

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