Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Key role of uridine kinase and uridine phosphorylase in the

homeostatic regulation of purine and pyrimidine

salvage in brain

Francesco Balestri

, Catia Barsotti

, Ludovico Lutzemberger

,

Marcella Camici

, Piero Luigi Ipata

*

a_{Dipartimento di Biologia, Unita` di Biochimica, Universita` di Pisa, Via S. Zeno 51, 56100 Pisa, Italy} b_{Dipartimento di Neuroscienze, Sezione di Neurochirurgia, Universita` di Pisa, Via Roma 55, 56100 Pisa, Italy}

Received 30 March 2007; received in revised form 12 June 2007; accepted 14 June 2007 Available online 22 June 2007

Abstract

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken

into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first

step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to b-alanine in liver. In the

present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain,

which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple

precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation

of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage

pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine

salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP

level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine

kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Salvage pathways; Uridine kinase; Uridine phosphorylase; Purine and pyrimidine homeostasis; Ribose-1-phosphate; 5-Phosphoribosyl-1-pyropho-sphate

1. Introduction

In humans, the predominant circulating pyrimidine is

uridine (Wu et al., 1994; Cansev, 2006). Among different

species, including man, its plasma level is strictly maintained at

3–5 mM, a concentration higher than that of other nucleosides

(Traut, 1994). Uridine is an important precursor of pyrimidine

salvage pathway in many tissues and cultivated cells

(Shambaugh, 1979; Karle et al., 1984; Moyer and Henderson,

1985; Traut and Jones, 1996; Barsotti et al., 2002) and

participates in the regulation of a number of physiological

processes especially in the central nervous system (Cansev,

2006, for a review). In normal conditions uridine, rather than

cytidine, is the major precursor for endogenous CDP-choline

production in rat, because uridine is transported more

efficiently than cytidine (Cansev, 2006). Moreover, uridine

acts as a physiological regulator of sleep function (Honda et al.,

1985; Inoue et al., 1995; Cao et al., 2005). With these

physiological roles in mind, it is no surprise that uridine has

found several clinical applications in the treatment of autism

with seizures (Page et al., 1997) and in genetic diseases, such as

orotic aciduria (Becroft et al., 1969). In cancer chemotherapy

based on pyrimidine antimetabolites, RNA, damaged by

fluoropyrimidine incorporation, can be rescued by exogenous

uridine administration (Darnowski et al., 1991; Pizzorno et al.,

1992). Finally, during severe hypoglycemia and ischemia,

uridine demonstrates to maintain brain metabolism (Dagani

et al., 1984; Pizzorno et al., 2002).

www.elsevier.com/locate/neuint

* Corresponding author. Tel.: +39 0502211452; fax: +39 0502211460. E-mail address:plipata@biologia.unipi.it(P.L. Ipata).

0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2007.06.007

In 2005,

Cao et al. (2005)

showed that disruption of uridine

homeostasis by deletion of rat uridine phosphorylase gene leads

to disorders of both pyrimidine and purine nucleotide synthesis,

thus suggesting a linkage between the two metabolic processes.

In this paper we present evidence that in brain, which relies

on the salvage of preformed purine and pyrimidine rings for

nucleotide synthesis, the metabolic sensor which maintains this

balance between the two salvage processes is a two enzyme

system, composed of uridine kinase (UK) and uridine

phosphorylase (UPase) (Fig. 1). The experiments were

performed by using rat brain extracts and a human astrocytoma

cell line (ADF). When UK, which catalyzes an irreversible

reaction, is inhibited by UTP and CTP concentrations

exceeding the physiological levels, the rate of pyrimidine

salvage lowers, uridine accumulates, and the equilibrium of the

reversible UPase reaction is shifted towards uridine

phosphor-olysis.

Formation

of

5-phosphoribosyl-1-pyrophosphate

(PRPP) from ribose-1-phosphate (Rib-1-P) would then favor

purine salvage. When UK is active, uridine is channelled

predominantly towards pyrimidine nucleotide synthesis, thus

lowering the rate of purine salvage synthesis.

2. Materials and methods

2.1. Materials

[2-14C]Uridine (53 mCi/mmol) was from Moravek Biochemicals (Brea, CA, USA). [2-14C]Uracil (54 mCi/mmol), [8-14C]adenine (55 mCi/mmol), [2-14C]5-fluorouracil (5-FU) (53 mCi/mmol), bases, nucleosides, and nucleo-tides were purchased from Sigma (St. Louis, MO, USA). HiSafe II scintillation liquid was purchased from LKB Pharmacia (Uppsala, Sweden). Polyethyle-neimmine (PEI)-cellulose precoated thin-layer plastic sheets (0.1 mm thick) were purchased from Merck (Darmstadt, Germany) and prewashed once with 10% NaCl and three times with deionized water before use. RPMI medium 1640, Dulbecco’s modified Eagle medium without glucose (DMEM) and foetal bovine serum (FBS) were from Gibco (Berlin, Germany). Human astrocytoma cells (ADF) were a kind gift of Dr. W. Malorni, Istituto Superiore di Sanita` (Roma, Italy). All other chemicals were of reagent grade.

2.2. Preparation of rat brain extract

Three-month-old male Sprague–Dawley rats (250 g) were sacrificed by decapitation. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policy. The brain was removed and kept frozen at 80 8C until needed. The post-mitochondrial extract, devoid of plasma mem-branes, nuclei, and mitochondria, was prepared as previously described ( Bar-sotti et al., 2002). Briefly, the brain was cut into small pieces, washed with cold saline and gently homogenized with a hand-driven Potter homogenizer in three volumes of 100 mM Tris–HCl buffer pH 7.4, with 20 mM KCl and 1 mM dithiothreitol (DTT). The homogenate was centrifuged at 4 8C at 39,000 g for 1 h. The supernatant fluid obtained was dialyzed overnight at 4 8C in dialysis bags against 10 mM Tris–HCl buffer, pH 7.4, supplemented with 1 mM DTT, and is referred to as crude extract. Protein concentration was determined by the Coomassie blue binding assay, using bovine serum albumin as standard (Bradford, 1979).

2.3. Preparation of human brain extracts

A single apparently undamaged sample of human frontal lobe was removed under the usual brain surgery conditions for tumor removal in the surgical room,

in agreement with the ethical rules for using human tissues for medical research in Italy. The sample, weighing about 3 mg, was immediately homogenized in about three volumes of 100 mM Tris–HCl buffer, pH 7.4, and treated as described in the previous section for rat brain. The final crude brain extract preparation contained 3.1 mg protein/ml.

2.4. Incubation procedures with post-mitochondrial rat brain

extracts

To follow purine salvage synthesis, the standard reaction mixture contained 50 mM Tris–HCl buffer, pH 7.4, 50 mM [8-14_{C]adenine (15,000 dpm/nmol),}

50 mM uridine, 3.6 mM ATP, 0.2 mM CTP, 0.2 mM UTP, 0.1 mM MnCl2,

8.3 mM MgCl2, 5 mM KH2PO4, and rat brain extract (1.15 mg protein/ml). To

follow pyrimidine salvage synthesis, the same reaction mixture was used, containing cold adenine and radioactive uridine (15,000 dpm/nmol). Modifica-tions of the standard incubation mixture are indicated in the figure legends. The reaction was started by the addition of crude extract. At different time intervals, the reaction was stopped by rapidly drying portions of 10 ml of the incubation mixture on PEI-cellulose precoated thin-layer plastic sheets and a chromato-gram was developed overnight with n-propanol/TCA (100% saturation)/NH3/

H2O (75/0.7/5/20, v/v). In all separations, appropriate standards were used and

detected as ultraviolet absorbing areas. The zones corresponding to radioactive UMP and UDP + UTP (total uracil nucleotides, TUN), to radioactive uracil, and to radioactive AMP and ADP + ATP (total adenine nucleotides, TAN) were excised and counted for radioactivity with 8 ml of scintillation liquid.

2.5. Human brain UPase assay

Human brain UPase phosphorolytic activity was determined by incubating brain extract (1.5 mg protein/ml) with 1 mM [2-14C]uridine (15,000 dpm/ nmol), and 50 mM Tris–HCl buffer, pH 7.4, containing 5 mM KH2PO4. The

reaction was started by addition of crude extract. Human brain UPase ribosylat-ing activity was determined by incubatribosylat-ing brain extract (1.5 mg protein/ml) with 1 mM [2-14C]uracil (15,000 dpm/nmol), 1 mM Rib-1-P, and 50 mM Tris– HCl buffer, pH 7.4. The reaction was started by addition of crude extract. The reaction mixtures were incubated at 37 8C. At different time intervals, the reaction was stopped by rapidly drying portions of 10 ml of the incubation mixture on PEI-cellulose precoated thin-layer plastic sheets and a chromato-gram was developed in 0.1 M (NH4)2SO4. The marker compounds were

detected under a UV lamp. The zones corresponding to uracil and uridine were excised and counted for radioactivity with 8 ml of scintillation liquid.

2.6. Incubation of human astrocytoma cells with purine and

pyrimidine compounds

Cells were routinely grown in RPMI medium with 10% FBS and 2% antibiotics at 37 8C in a humidified 5% CO2/95% air atmosphere. The

experi-ments were performed with cells (approximately 1,500,000) kept in 35-mm plates for different times in 0.5 ml of serum-free DMEM medium. In order to test the purine salvage synthesis, cells were incubated for the indicated times with 50 mM [8-14_{C]adenine (15,000 dpm/nmol) alone or in the presence of}

either 100 mM inosine or deoxyinosine, or uridine (from 50 to 200 mM); for pyrimidine salvage, cells were incubated with 50 mM [2-14_C]uracil

(35,000 dpm/nmol) alone or in the presence of 100 mM inosine, or with 100 mM [2-14C]uridine (5600 dpm/nmol) alone or in the presence of 50 mM adenine. The formation of 5-FU nucleotides was followed by incubating 50 mM [2-14C]5-FU (10,500 dpm/nmol) either alone or in the presence of 100 mM inosine. At the different times of incubation, the medium was removed, and cells were washed twice with 2 ml of cold physiological solution. For the extraction of purine and pyrimidine compounds, 0.1 ml of ice-cold 0.6 M perchloric acid were added to each plate and kept for 15 min; cells were harvested with a cell scraper, and the suspension was centrifuged in an Eppendorf Microfuge. The supernatant was neutralized with 15 ml of 3.5 M K2CO3. After centrifugation in a Microfuge, 50 ml of the supernatant were

applied to a PEI-cellulose plate which was developed overnight with n-propanol/TCA (100% saturation)/NH3/H2O (75/0.7/5/20, v/v). The zones

radioactive UMP + UDP + UTP (TUN) (uracil and uridine salvage) or F-UMP + F-UDP + F-UTP (5-FU activation) were cut and the radioactivity counted, after addition of 8 ml of liquid scintillation counter.

3. Results

Tha rationale of our experimental procedure is the

following. Since the purine and pyrimidine salvage processes

occur at the base and nucleoside level, respectively (Murray,

1971; Kim et al., 1992; Barsotti et al., 2002; Lo¨ffler et al., 2005)

rat brain extract or cultured human astrocytoma cells (ADF) are

incubated in the presence of either radioactive uridine and cold

adenine, to follow the rate of pyrimidine salvage, or with cold

uridine and radioactive adenine, to follow the rate of purine

salvage. We emphasize that this experimental procedure allows

us to measure the rate of purine salvage when also pyrimidine

Fig. 2. Effect of different ATP, UTP, and CTP concentrations on the metabolic fate of uridine and adenine in rat brain extracts. (A) The incubation mixtures contained 50 mM adenine and 50 mM [2-14C]uridine (15,000 dpm/nmol) (open symbols), or 50 mM [8-14C]adenine (15,000 dpm/nmol) and 50 mM uridine (closed symbols), 3.6 mM ATP, 0.2 mM UTP, 0.2 mM CTP, 0.1 mM MnCl2, 8.3 mM MgCl2, 5 mM KH2PO4, 50 mM Tris–HCl buffer, pH 7.4, and rat brain extract (1.15 mg protein/

ml). (B) The incubation mixtures were as above, but contained 1 mM ATP, 2 mM UTP, and 2 mM CTP. (*) Total uracil nucleotide (TUN); (*) total adenine nucleotide (TAN).

Fig. 1. Interrelationship between purine and pyrimidine salvage. The enzymes participating in the pathway are: (1) purine nucleoside phosphorylase; (2) UPase; (3) UK; (4) nucleoside-monophosphate kinase; (5) nucleoside-diphosphate kinase; (6) phosphopentomutase; (7) PRPP synthetase; (8) phosphoribosyltransferases. ATP is a substrate of UK, nucleoside-monophosphate, and nucleoside-diphosphate kinases, while UTP and CTP are specific inhibitors of UK. At high [ATP]/ [UTP] + [CTP] ratio, a signal of purine nucleotide sufficiency and pyrimidine nucleotide insufficiency, the flux towards pyrimidine salvage is favored, because uridine is continuously and irreversibly phosphorylated by the active UK. Moreover, in these conditions, some uracil (and 5-FU) might be anabolized to their respective nucleotides, in the presence of Rib-1-P. Conversely, at low [ATP]/[UTP] + [CTP] ratio, the metabolic flux towards purine salvage is favored, because UK becomes inhibited, and the equilibrium of UPase is shifted towards uracil and Rib-1-P. Urd: uridine; Ura: uracil; Ino: inosine; Guo: guanosine; Hyp: hypoxanthine; Gua: guanine; Ade: adenine. ( ) Inhibition.

salvage is proceeding and vice versa. The time course of

radioactive purine and pyrimidine nucleotide formation is

followed. The results will be discussed with the aid of

Fig. 1,

which summarizes our present knowledge on the interplay

between purine and pyrimidine salvage.

Fig. 2

shows the effect

of two different mixtures of ATP, UTP, and CTP on the time

course of purine and pyrimidine salvage processes, catalyzed

by rat brain post-mitochondrial extract. We recall that ATP acts

as phosphate donor in nucleoside, nucleoside-monophosphate,

and nucleoside-diphosphate kinases, as well as the PPi donor to

Rib-5-P in PRPP synthesis, with K

values in the high

micromolar range (Kornberg et al., 1955; Kimura and Shimada,

1988; Van Rompay et al., 2001; Pasti et al., 2003). It can be seen

that the two salvage processes respond in an opposite manner.

Thus, at ATP, UTP, and CTP concentrations of 3.6 mM,

0.2 mM, and 0.2 mM, respectively, the rate of pyrimidine

salvage was much higher than that of purine salvage. However,

at 1 mM ATP and at UTP and CTP each at 2 mM, as expected,

the pyrimidine salvage was virtually fully inhibited, but at the

same time a higher purine salvage rate was observed. The

results presented in

Fig. 3

clearly show that the amount of

pyrimidine and purine nucleotides formed are a direct and an

indirect function of the [ATP]/[UTP] + [CTP] ratio,

(TAN) formation as a function of [ATP]/[UTP] + [CTP] ratio. The incubation mixtures contained 50 mM adenine and 50 mM [2-14C]uridine (15,000 dpm/ nmol) (* TUN, and & uracil), or 50 mM [8-14C]adenine (15,000 dpm/nmol) and 50 mM uridine (* TAN), 0.1 mM MnCl2, 8.3 mM MgCl2, 5 mM KH2PO4,

50 mM Tris–HCl buffer, pH 7.4, and rat brain extract (1.15 mg protein/ml). The amount of uracil, TAN, and TUN formed after 30 min incubation is reported. The total final concentration of added nucleoside triphosphates was always 4 mM; UTP and CTP were added each at the same final concentration. For instance, at the ratio of 4, ATP, UTP, and CTP concentrations were 3.2 mM, 0.4 mM and 0.4 mM each, while at the ratio of 9, ATP, UTP, and CTP concentrations were 3.6 mM, 0.2 mM, and 0.2 mM each.

Fig. 4. Time course of total adenine nucleotide (TAN) formation from adenine in cultured human astrocytoma cells. ADF cells were incubated with 50 mM [8-14C]adenine (15,000 dpm/nmol) in the absence (*) and in the presence of 100 mM inosine (*), or 100 mM uridine (&), or 100 mM deoxyinosine (&). The inset shows the rate of TAN formation from adenine at the indicated concentrations of uridine.

Fig. 5. Time course of total uracil nucleotide (TUN) formation from uridine in cultured human astrocytoma cells. ADF cells were incubated with 100 mM [2-14_{C]uridine (5600 dpm/nmol) either in the absence (*) or in the presence}

(*) of 50 mM adenine.

Fig. 6. Time course of total uracil nucleotide (TUN) formation from uracil and of 5-FU nucleotide formation from 5-FU in cultured human astrocytoma cells. ADF cells were incubated with 50 mM [2-14C]uracil (35,000 dpm/nmol) (open symbols) or with 50 mM [2-14C]5-FU (10,500 dpm/nmol) (closed symbols) in the absence (*, *) and in the presence of 100 mM inosine (&, &).

tively. It also shows that the highest amount of uracil formed at

the lowest ratio. It has long been known that uridine can be

taken up by intact cells, and salvaged to uridine nucleotides

(Rozengurt et al., 1978; Cansev, 2006). Exogenous uridine and,

to a lesser extent inosine, activate the salvage of exogenous

adenine in intact cultured human astrocytoma cells (ADF) in a

concentration-dependent manner (Fig. 4).

Fig. 5

shows that the

transformation of exogenous uridine into endogenous uracil

nucleotides in ADF cells is consistently lowered by

exogen-ously added adenine. Moreover, exogenous inosine favors 5-FU

activation and uracil salvage (Fig. 6).

The distribution of active UPase in human tissues is a

matter of some debate (Lo¨ffler et al., 2005). Since UPase is

essential in the mechanism of reciprocal regulation of purine

and pyrimidine salvage in rat brain, its activity was measured

in a cytosolic preparation of human brain. Specific activities of

1.02 nmol uracil formed/(mg protein min) and 0.18 nmol

ur-idine formed/(mg protein min) were found for the

phosphor-olytic and ribosylating reaction, respectively. These results

are in agreement with those of

Pizzorno et al. (2002), who

reported UPase activity in homogenates of a list of human

tissues and from tumors, and with those of

Balestri et al.

(2007)

who reported that ADF cells also exhibit UPase

activity.

4. Discussion

It is generally accepted that only liver and kidney maintain

the de novo pyrimidine and purine synthesis and supply other

tissues and organs, including brain, with preformed pyrimidine

nucleosides (mainly uridine) and purine nucleosides and bases

for nucleotide synthesis (Barsotti et al., 2002; Cao et al., 2005;

Cansev, 2006). This raises the following question: how do these

districts, which rely more heavily on salvage synthesis,

maintain the right balance between the purine and pyrimidine

pools for the stability of genetic information? In this regard, we

have hypothesized that the UPase–UK enzyme system, which

maintains uridine homeostasis, regulates the two processes of

purine and pyrimidine salvage. The kinase is the major entry

step in the salvage of preformed uridine to UTP, and then to

CTP, while the phosphorylase is the major entry step in the

catabolism of uridine to b-alanine, a process considered to be

restricted to the liver (Reichard and Sko¨ld, 1958; Connolly and

Duley, 1999; Lo¨ffler et al., 2005). UK is inhibited by elevated

UTP and CTP levels, a signal of pyrimidine sufficiency, and

activated by ATP, a signal of purine sufficiency (Cheng et al.,

1986; Ropp and Traut, 1998; Suzuki et al., 2004). This kind of

regulation is reminescent of that exerted by UTP and CTP,

acting as inhibitors, and ATP, acting as activator, on

carbamoyl-phosphate synthetase and aspartate transcarbamylase, the

committed steps of de novo pyrimidine biosynthesis in

mammalian and bacterial cells, respectively (Jones, 1980;

Allewell, 1989; England and Herve, 1994). The results

presented in

Fig. 2

may be explained by admitting that when

the concentration of UTP and CTP is relatively high, the

inhibition of UK causes a shift of the equilibrium of the

reversible UPase reaction towards uridine phosphorolysis. The

Rib-1-P formed is then converted into PRPP, which is used in

the salvage synthesis of purine nucleotides (Wice and Kennel,

1982; Inoue et al., 1995). Conversely, when the concentration

of UTP and CTP is relatively low, the fully active UK, which

catalyzes a virtual irreversible reaction, drives uridine towards

uridine nucleotide formation, thus lowering the rate of purine

synthesis (see

Fig. 2). Even though concentrations of UTP and

CTP as high as 2 mM each used in

Fig. 2B may not be reached

in vivo, the results suggest that purine and pyrimidine salvage

might be reciprocally regulated in rat brain, simply by

transmitting to UPase the modulation of UK by UTP and

CTP. Therefore, the effect of different [ATP]/[UTP] + [CTP]

ratios on purine and pyrimidine salvage was investigated, by

adding ATP, UTP, and CTP, each at reasonable ‘‘physiological’’

concentrations (Traut, 1994). The results presented in

Fig. 3

clearly show that the amount of pyrimidine and purine

nucleotides formed are strictly related to the value of the

[ATP]/[UTP] + [CTP] ratio. In addition,

Fig. 3

shows that the

highest amount of uracil formed at the lowest ratio, when UK is

inhibited.

Since [ATP]/[UTP] + [CTP] ratios between 5 and 9, when

both purine and pyrimidine salvage are operative in vitro

(Fig. 3), reasonably correspond to physiological fluctuation of

the three nucleotides (Traut, 1994), we reasoned that in well

oxygenated intact cells exogenous uridine, which is readily

uptaken by cultured cells (Griffith and Jarvis, 1996; Nagai

et al., 2005; Cansev, 2006) might favor purine salvage. As

shown in

Fig. 4

in cultured ADF cells, exogenous uridine

stimulates adenine salvage in a concentration-dependent

manner. It may be speculated that as uridine accumulates,

also in view of the inhibition exerted on UK by the raise of

pyrimidine nucleotides (see also

Fig. 5), more Rib-1-P

becomes available, through the action of UPase, for the

PRPP-mediated adenine salvage. In this regard, also inosine,

which, through purine nucleoside phosphorylase, acts as

Rib-1-P donor, appears to stimulate adenine salvage, while

deoxyinosine is without effect. Since deoxyRib-5-P is not a

substrate of PRPP synthetase, the lack of effect of

deox-yinosine is a further indication that the salvage of the purine

ring occurs at the nucleobase level, using PRPP as the donor of

the phosphoribosyl moiety. In addition, the increased

utiliza-tion of PRPP exerted by added adenine, might further favor

uridine phosphorolysis, thus explaining the apparent inhibition

exerted by adenine on exogenous uridine salvage to uracil

nucleotides (Fig. 5). We emphasize that adenine, even at 1 mM,

does not inhibit uridine transport (Crawford et al., 1998). On

the other hand, we have also demonstrated that the purine

inosine stimulates 5-FU activation and uracil salvage (Fig. 6):

most likely, this occurs through a Rib-1-P-mediated process,

because uracil phosphoribosyltransferase is absent in

mam-mals (Traut and Jones, 1996; Cappiello et al., 1998). However,

it cannot be excluded a priori that 5-FU activation might occur

through the PRPP-mediated process, catalyzed by orotate

phosphoribosyltransferase acting on 5-FU (Mascia and Ipata,

2001).

We propose the following stoichiometry for the

intercon-nected pyrimidine and purine salvage processes.

The observations that the mammalian brain relies on a

supply of circulating purines and pyrimidines, such as inosine,

guanosine, and uridine for energy repletion and phospholipid

synthesis (Shambaugh, 1979; Moyer and Henderson, 1985;

Gonzalez and Fernandez-Salguero, 1995; Connolly et al., 1996;

Jurkowitz et al., 1998; Connolly and Duley, 1999; Barsotti

et al., 2002; Balestri et al., 2007) emphasize the importance of

purine and pyrimidine nucleoside salvage in the maintenance of

CNS activity. Moreover, understanding the process that

regulates the intracellular nucleotide homeostasis will enhance

our ability to manipulate salvage pathways for chemotherapy

(Natsumeda et al., 1989; Mascia and Ipata, 2001). In this paper

we have shown that the two processes of purine and pyrimidine

salvage are regulated at the level of UPase–UK enzyme system

by the relative purine nucleoside triphosphate/pyrimidine

nucleoside triphosphate concentration. Further studies are

needed to clarify the role of different brain cell types in this

regulatory process, also in view of the recent finding that

uridine is more efficiently taken up by neurons than by

astrocytes (Nagai et al., 2005).

Acknowledgements

This work was supported by a grant from Italian MIUR

(National Interest Project: ‘‘Molecular mechanisms of cellular

and metabolic regulation of polynucleotides, nucleotides, and

analogs’’) and local funds from University of Pisa.

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